Isolation and characterization of polymorphic microsatellite DNA markers in two shrew species,Sorex unguiculatus and S. caecutiens

We isolated six microsatellite markers from the partial genomic libraries of two Sorex shrews, S. unguiculatus and S. caecutiens, and examined their allelic variation. All loci showed high allelic variation ranging from 15 to 19 alleles and all but one locus conformed to Hardy–Weinberg expectations in the species where the loci were isolated. Cross-species amplifications showed that all primers derived from S. unguiculatus were useful for S. caecutiens, while among primer sets derived from S. caecutiens only one was useful for S. unguiculatus. Accordingly, at least five microsatellite markers were useful in S. caecutiens and three in S. unguiculatus.     

Population genetic structure of Sorex unguiculatus and Sorex caecutiens (Soricidae, Mammalia) in Hokkaido, based on microsatellite DNA polymorphism

We investigated the genetic structure of Sorex unguiculatus and Sorex caecutiens populations in Hokkaido, Japan, using hypervariable microsatellite DNA markers. We used five microsatellite loci to type 475 S. unguiculatus individuals from 20 localities on the Hokkaido mainland and four localities from each of four offshore islands (and 11 shrews from one locality in southern Sakhalin for a particular analysis). We used six microsatellite loci to type 240 S. caecutiens individuals from 13 localities on the Hokkaido mainland. Genetic variation was high in mainland populations of both species and low in the island populations of S. unguiculatus. Allelic richness and island size were positively correlated for S. unguiculatus, suggesting that genetic drift occurred on those islands due to small population size. In addition, four insular populations of S. unguiculatus were genetically differentiated from the mainland populations, although clear phylogeographic clustering was not confirmed among populations on the Hokkaido mainland for either S. unguiculatus or S. caecutiens. Heterozygosity excess was observed in more than half of the populations including the mainland populations of the two species, suggesting recent bottleneck events in these populations. Population dynamics of the shrews might be explained by a metapopulation scheme. According to autocorrelation analysis, the extent of non-random spatial genetic structure was approximately 100 km. Isolation by distance was observed in S. unguiculatus, but not in S. caecutiens although there is a positive trend. The lack of correlation for S. caecutiens might have been due to small sample size. Thus, no obvious differences in population genetic structure were found between the two species on the Hokkaido mainland in the present study, while previous investigations using mitochondrial DNA sequences inferred that these two species might have rather different biogeographic histories.     

Sorex rohweri sp. nov. (Mammalia, Soricidae) from northwestern North America

Sorex rohweri sp. nov. is described on the basis of a series of specimens from the Olympic Peninsula and adjacent western regions of Washington State, USA, and southwestern British Columbia, Canada. It has been misidentified as Sorex cinereus Kerr, 1792, which occurs in the Cascade Range in west-central Washington, in coastal British Columbia, and regions farther to the northeast. The new species is distinguished from S. cinereus by numerous morphological characters: differences in craniodental dimensions; different location and form of the medial tines of the upper incisors; presence of patent postmandibular foramina; different intensity and distribution of dental pigmentation; and different form of the glans penis, along with other details. The combination of characters also separates it from sympatric species of Sorex. Phylogenetic inference of cytochrome b sequences from two specimens of the proposed new species shows them to be distinct from Sorex cinereus and from five sympatric Sorex species, supporting their designation as members of a new species.     

A multiplex PCR assay for molecular sexing of the naked mole-rat (Heterocephalus glaber)

For molecular sexing of the naked mole-rat (Heterocephalus glaber), we designed a PCR primer set to amplify part of the Y-linked DBY gene. When this primer set was applied to the samples of known sex with the 16S rRNA gene (16S rDNA) primers as control, PCR products were successfully obtained as two DNA bands in males, a male-specific 163 bp DBY band and a 446 bp band of 16S rDNA shared with females, whereas females showed only the common band. This result shows that this multiplex PCR assay is useful for sex identification of H. glaber.     

Environmental DNA enables detection of terrestrial mammals from forest pond water

Terrestrial animals must have frequent contact with water to survive, implying that environmental DNA (eDNA) originating from those animals should be detectable from places containing water in terrestrial ecosystems. Aiming to detect the presence of terrestrial mammals using forest water samples, we applied a set of universal PCR primers (MiMammal, a modified version of fish universal primers) for metabarcoding mammalian eDNA. The versatility of MiMammal primers was tested in silico and by amplifying DNAs extracted from tissues. The results suggested that MiMammal primers are capable of amplifying and distinguishing a diverse group of mammalian species. In addition, analyses of water samples from zoo cages of mammals with known species composition suggested that MiMammal primers could successfully detect mammalian species from water samples in the field. Then, we performed an experiment to detect mammals from natural ecosystems by collecting five 500‐ml water samples from ponds in two cool‐temperate forests in Hokkaido, northern Japan. MiMammal amplicon libraries were constructed using eDNA extracted from water samples, and sequences generated by Illumina MiSeq were subjected to data processing and taxonomic assignment. We thereby detected multiple species of mammals common to the sampling areas, including deer (Cervus nippon), mouse (Mus musculus), vole (Myodes rufocanus), raccoon (Procyon lotor), rat (Rattus norvegicus) and shrew (Sorex unguiculatus). Many previous applications of the eDNA metabarcoding approach have been limited to aquatic/semiaquatic systems, but the results presented here show that the approach is also promising even for forest mammal biodiversity surveys.     

PCR identification of the host DNA in the taiga tick (Ixodinae: <Emphasis Type="Italic">Ixodes persulcatus</Emphasis>) nymphs in St. Petersburg and its environs

Host DNA from hungry males and females of Ixodes persulcatus (a total of 143 specimens) collected in the wild in April–May 2008 and 2009 was identified by means of the PCR method. Amplification of each sample was performed using a primer species-specific by the 12S rRNA mitochondrial gene. Four species of small mammals (Apodemus uralensis, Clethrionomys glareolus, Microtus arvalis, and Sorex araneus) and two species of passerine birds (Fringilla coelebs and Parus major) were analyzed. Hosts were established for 44 samples (30.8% of the material); in five cases (3.5%), feeding on more than one host was revealed.     

Species- and sex-specific multiple pcr amplifications of partial cytochromeb gene andZfx/Zfy introns from invasive and non-invasive samples of Korean ungulates

There are five representative ungulates (e.g., goral,Nemorhaedus caudatus; goat,Capra hircus; roe deer,Capreolus pygargus; water deer,Hydropotes inermis; musk deer,Moschus moschiferus) in the wild of South Korea. Their fecal morphologies are similar to each other between species or sexes, and therefore it is very difficult to identify the species or sex. To distinguish the species and sex of the elusive animals, we developed species- and sex-specific multiple PCR amplifications using pairs of primer sets. Partial cytochromeb gene andZfx/Zfy introns were targeted for the PCR amplifications. For species identification using a multiple primer set (BJGL-F1/GT-F1/RD-F1/WD-F1/MD-F1 and BJGL-R/GT-R/RD-R/WD-R /MD-R), all (n=21/21) of the invasive samples were correctly identified. About 71% (n=15/21) of the non-invasive samples were successfully identified. The multiple primer set could determine each species which showed a unique sized PCR fragment. For sex identification using a multiple primer set (BJSexX-F/Y-F and BJSex-R1), about 90% (n=19/21) of invasive samples were correctly determined. About 62% (n=13/21) of the non-invasive samples were successfully identified. Using the multiple primer set, both X-and Y-chromosome linked bands for males (n=21) and only X-chromosome linked band for females (n=11) could be detected. The results would be applicable to the species and sex identification of the Korean and the Far-East Asian wild ungulates.     

Overlap of temporal niches among four sympatric species of shrews

Hypotheses about the dependence of circadian activity from metabolic rate and the segregation of temporal niches among competing species were verified by the study of activity patterns in a shrew community of two semiaquatic species,Neomys anomalus Cabrera, 1907 andN. fodiens (Pennant, 1771), and two terrestrial species,Sorex araneus Linnaeus, 1758 andS. minutus Linnaeus, 1766, co-existing in wet habitats of Białowieża Forest (E Poland). In ten trapping sessions, performed in early summer between 1991 and 2000, traps were open 24 hours continuously and patrolled at 1:00, 5:00, 10:00, 15:00, and 20:00. All the shrew species were most active between 20:00 and 1:00, and least active around mid-day (10:00–15:00). However, activity of the twoSorex species was lower than that of the twoNeomys species in the period 20:00–1:00, but higher in the period 15:00–20:00. BothNeomys species displayed clearly nocturnal, unimodal patterns of activity. In contrast, activity of bothSorex species was relatively evenly distributed over 24 hours and they increased their activity earlier (ie after 15:00) than bothNeomys species (after 20:00). These results confirm the idea that small shrew species with higher metabolic rate have more frequent and more equally distributed activity bouts than large species. Overlap of temporal niches was the highest within genera (99.29% between bothNeomys species and 98.36% between bothSorex species), the lowest betweenN. fodiens andS. araneus (88.26%) andS. minutus (89.34%), and intermediate betweenN. anomalus and bothSorex species (91.78 and 93.34%, respectively). Such high interspecific overlaps in activity suggest a joint-action of other mechanisms that separate ecological niches of these species also in other dimensions (eg food, microhabitat).     

Sex Identification of Four Penguin Species Using Locus‐Specific PCR

Traditional methods for sex identification are not applicable to sexually monomorphic species, leading to difficulties in the management of their breeding programs. To identify sex in sexually monomorphic birds, molecular methods have been established. Two established primer pairs (2550F/2718R and p8/p2) amplify the CHD1 gene region from both the Z and W chromosomes. Here, we evaluated the use of these primers for sex identification in four sexually monomorphic penguin species: king penguins (Aptenodytes patagonicus), rockhopper penguins (Eudyptes chrysocome), gentoo penguins (Pygoscelis papua), and Magellanic penguins (Spheniscus magellanicus). For all species except rockhopper penguins, primer pair 2550F/2718R resulted in two distinct CHD1Z and CHD1W PCR bands, allowing for sex identification. For rockhopper penguins, only primer pair p8/p2 yielded different CHD1Z and CHD1W bands, which were faint and similar in size making them difficult to distinguish. As a result, we designed a new primer pair (PL/PR) that efficiently determined the gender of individuals from all four penguin species. Sequencing of the PCR products confirmed that they were from the CHD1 gene region. Primer pair PL/PR can be evaluated for use in sexing other penguin species, which will be crucial for the management of new penguin breeding programs. Zoo Biol 32:257–261, 2013. © 2012 Wiley Periodicals, Inc.     

The karyotype of the Azumi shrew<Emphasis Type="Italic">Sorex hosonoi</Emphasis>

Karyotype ofSorex hosonoi Imaizumi, 1954 from Mt. Asama in central Honshu, Japan, were examined with conventional staining and G-banding using ASG methods. The diploid and fundamental autosomal arm numbers were 42 and 66, respectively. The autosomes consisted of seven metacentric, six submetacentric, and seven acrocentric pairs. The sex chromosomes were large-sized acrocentric X and small-sized subtelocentric Y. The relationship between the karyotypes ofS. hosonoi andS. shinto was explained by one pericentric inversion at the no. 5 ofS. hosonoi and the no. 9 ofS. shinto. A rearrangement inS. shinto-hosonoi differed from the rearrangements occurring on no. 5 ofS. shinto-caecutiens/unguiculatus.     

Structure of the sinus hair follicle in the big-clawed shrew,Sorex unguiculatus

The structural features of sinus hair follicles in Sorex unguiculatus were studied by macroscopic dissection, serial section light microscopy and electron microscopy. The shrew has about 540 sinus hairs regularly arranged on the snout. The maxillary nerves innervating them are extremely thick, while the optic nerves are very thin. Thus the follicle must be one of the most important sense organs in this animal. In the follicle the ring sinus is well-developed and the trabeculae of the cavernous sinus are reduced in number and thickness. The ring bulge is not a unified structure but a pair of bodies which consist of head, stalk and attachment plaque. It is characterized by the presence of numberous thick collagen fibrils (400 nm) and appears to be mechanically rigid. Lanceolate nerve terminals, free endings, Merkel cells with nerve terminals and unmyelinated fibers are observed, but encapsulated endings are lacking in and around the follicles. Straight lanceolate terminals on the posterior side of the follicle are thick and three-sided in cross section, while those on the anterior side are thin and two-sided. Free endings are located on the anterior side of the follicle. These and other findings are discussed on the basis of the assumption that the Sorex sinus hair follicle is more specialized as a vibrating system than in other mammals.     

Evolutionary and taxonomic differentiation of shrew species in the “araneus” group of the genus <Emphasis Type="Italic">Sorex</Emphasis>: 2. Subdivision within the common shrew

This review summarizes available data on the problem of taxonomic and evolutionary differentiation in the “araneus” groups of species of the genus Sorex (Eulipotyphla, Mammalia). Report 2 describes the hierarchical structuring, population system, and interracial hybrid zones in the common shrew (Sorex araneus).     

A novel method of caenophidian snake sex identification using molecular markers based on two gametologous genes

Sex identification provides important information for ecological and evolutionary studies, as well as benefiting snake conservation management. Traditional methods such as cloacal probing or cloacal popping are counterproductive for sex identification concerning very small species, resulting in difficulties in the management of their breeding programs. In this study, the nucleotide sequences of gametologous genes (CTNNB1 and WAC genes) were used for the development of molecular sexing markers in caenophidian snakes. Two candidate markers were developed with the two primer sets, and successfully amplified by a single band on the agarose gel in male (ZZ) and two bands, differing in fragment sizes, in female (ZW) of 16 caenophidian snakes for CTNNB1 and 12 caenophidian snakes for WAC. Another candidate marker was developed with the primer set to amplify the specific sequence for CTNNB1W homolog, and the PCR products were successfully obtained in a female‐specific 250‐bp DNA bands. The three candidate PCR sexing markers provide a simple sex identification method based on the amplification of gametologous genes, and they can be used to facilitate effective caenophidian snake conservation and management programs.     

The development and evaluation of consensus chloroplast primer pairs that possess highly variable sequence regions in a diverse array of plant taxa

Although universal or consensus chloroplast primers are available, they are limited by their number and genomic distribution. Therefore, a set of consensus chloroplast primer pairs for simple sequence repeats (ccSSRs) analysis was constructed from tobacco (Nicotiana tabacum L.) chloroplast sequences. These were then tested for their general utility in the genetic analysis of a diverse array of plant taxa. In order to increase the number of ccSSRs beyond that previously reported, the target sequences for SSR motifs was set at A or T (n 7) mononucleotide repeats. Each SSR sequence motif, along with ±200-bp flanking sequences from the first of each mononucleotide base repeat, was screened for homologies with chloroplast DNA sequences of other plant species in GenBank databases using BLAST search procedures. Twenty three putative marker loci that possessed conserved flanking sequence spans were selected for consensus primer pair construction using commercially available computer algorithms. All primer pairs produced amplicons after PCR employing genomic DNA from members of the Cucurbitaceae (six species) and Solanaceae (four species). Sixteen, 22 and 19 of the initial 23 primer pairs were successively amplified by PCR using template DNA from species of the Apiaceae (two species), Brassicaceae (one species) and Fabaceae (two species), respectively. Twenty of 23 primer pairs were also functional in three monocot species of the Liliaceae [onion (Allium cepa L.) and garlic (Allium sativum L.)], and the Poaceae [oat (Avena sativa L.)]. Sequence analysis of selected ccSSR fragments suggests that ccSSR length and sequence variation could be useful as a tool for investigating the genetic relationships within a genus or closely related taxa (i.e., tribal level). In order to provide for a marker system having significant coverage of the cucumber chloroplast genome, ccSSR primers were strategically "recombined" and named recombined consensus chloroplast primers (RCCP) for PCR analysis. Successful amplification after extended-length PCR of 16 RCCP primer pairs from cucumber (Cucumis sativus L.) DNA suggested that the amplicons detected are representative of the cucumber chloroplast genome. These RCCP pairs, therefore, could be useful as an initial molecular tool for investigation of traits related to a chloroplast gene(s) in cucumber, and other closely related species.Communicated by C. Möllers     

Characterization of six microsatellite DNA loci for Sorex arizonae

Sorex arizonae is a rare species that occupies a narrow range of habitat types in several mountain ranges of New Mexico, Arizona and Northern Mexico. Here we identify and characterize six microsatellite loci for this species. We screened 63 individuals from four different localities from New Mexico and Arizona to analyse genetic variability. Alleles ranged from three to 16. Heterozygosity ranged from 40% to 78%. Most polymorphic loci were in Hardy–Weinberg equilibrium with the exception of one locus. Primers appear to have reasonable cross‐species applicability as five loci amplified in another shrew species (Sorex monticolus).     

Evolutionary history of the genus Sorex (Soricidae,Eulipotyphla) as inferred from multigene data

The genus Sorex is one of the most diverse and ecologically successful lineages of the family Soricidae. We present the first multilocus nuclear phylogeny focusing on the nominal subgenus Sorex s.str., which is distributed mainly in the northern Palearctic. The nuclear tree (six exons) provides more resolution than the mitochondrial data (cytb) and supports subdivision into eight species groups within Sorex s.str., most of which correspond to those recognized from chromosome data. The European species S. alpinus is consistently placed as the basal lineage in the Palearctic clade, while the next split separates the east‐Tibetan group of striped shrews (S. aff. cylindricauda, S. bedfordiae, S. excelsus). Within the remaining species, the following well‐supported clades are identified at the supra‐group level: “araneus” species group+S. samniticus; the “caecutiens” group+the “minutus” group, the latter also including S. minutissimus, S. gracillimus and S. thibetanus. S. raddei and S. roboratus represent separate lineages with no close relatives. The fossil‐calibrated molecular clock placed the divergence between Sorex s.str. and Otisorex at the Early/Middle Miocene boundary. Basal radiation of the crown Sorex s.str. was estimated to have occurred in the middle of the Late Miocene. A more than threefold increase in the diversification rate is inferred for the Early Pliocene. Taxonomic implications including potential genus ranks for Sorex s.str. and Otisorex are discussed. S. alpinus is placed in the monotypic subgenus Homalurus. The full species status of S. buchariensis and S. thibetanus and close relationships between S. cf. cansulus and S. caecutiens are confirmed.     

A new marker based on the avian spindlin gene that is able to sex most birds,including species problematic to sex with CHD markers

We have developed a new marker (Z43B) that can be successfully used to identify the sex of most birds (69%), including species difficult or impossible to sex with other markers. We utilized the zebra finch Taeniopygia guttata EST microsatellite sequence (CK309496) which displays sequence homology to the 5′ untranslated region (UTR) of the avian spindlin gene. This gene is known to be present on the Z and W chromosomes. To maximize cross‐species utility, the primer set was designed from a consensus sequence created from homologs of CK309496 that were isolated from multiple distantly related species. Both the forward and reverse primer sequences were 100% identical to 14 avian species, including the Z chromosome of eight species and the chicken Gallus gallus W chromosome, as well as the saltwater crocodile Crocodylus porosus. The Z43B primer set was assessed by genotyping individuals of known sex belonging to 61 non‐ratite species and a single ratite. The Z and W amplicons differed in size making it possible to distinguish between males (ZZ) and females (ZW) for the majority (69%) of non‐ratite species tested, comprising 10 orders of birds. We predict that this marker will be useful for obtaining sex‐typing data for ca 6,869 species of birds (69% of non‐ratites but not galliforms). A wide range of species could be sex‐typed including passerines, shorebirds, eagles, falcons, bee‐eaters, cranes, shags, parrots, penguins, ducks, and a ratite species, the brown kiwi, Apteryx australis. Those species sexed include species impossible or problematic to sex‐type with other markers (magpie, albatross, petrel, eagle, falcon, crane, and penguin species). Zoo Biol. 35:533–545, 2016. © 2016 The Authors. Zoo Biology published by Wiley Periodicals, Inc.     

Synchrony between small mammal population dynamics in marshes and adjacent grassland in a landscape of the Jura plateau, France: a ten year investigation

Small mammal populations were studied during a 10 year multi-annual fluctuation of vole populations in marshes and adjacent grassland of the basin in the Jura mountains, France. Nine species were monitored:Microtus agrestis, Clethrionomys glareolus, Apodemus flavicollis, Sorex araneus Icoronatus, Neomys fodiens, Crocidura leucodon, Mustela nivalis in marshes andArvicola terrestris andMicrotus arvalis in grassland, using live-trapping and index methods during July from 1993 to 2002. Populations of A.terrestris, M. arvalis, M. agrestis, S. araneus I coronatus andM. nivalis exhibited a relatively strong inter-specific temporal synchrony in their multi-annual fluctuations.C. glareolus population fluctuations were not synchronous to theArvicola I Microtus I Sorex group, and were closer to those ofApodemus flavicollis, Neomys andCrocidura. Those results are discussed in the light of earlier studies carried out in the same region and in northern ecosystems.     

A Species-Specific Polymerase Chain Reaction Assay for Rapid and Sensitive Detection of <Emphasis Type="Italic">Colletotrichum</Emphasis><Emphasis Type="Italic">capsici</Emphasis>

Colletotrichum capsici is an important fungal species that causes anthracnose in many genera of plants causing severe economic losses worldwide. A primer set was designed based on the sequences of the ribosomal internal transcribed spacer (ITS1 and ITS2) regions for use in a conventional PCR assay. The primer set (CcapF/CcapR) amplified a single product of 394 bp with DNA extracted from 20 Mexican isolates of C. capsici. The specificity of primers was confirmed by the absence of amplified product with DNA of four other Colletotrichum species and eleven different fungal genera. This primer set is capable of amplifying only C. capsici from different contaminated tissues or fungal structures, thereby facilitating rapid diagnoses as there is no need to isolate and cultivate the fungus in order to identify it. The sensitivity of detection with this PCR method was 10 pg of genomic DNA from the pathogen. This is the first report of a C. capsici-specific primer set. It allows rapid pathogen detection and provides growers with a powerful tool for a rational selection of fungicides to control anthracnose in different crops and in the post-harvest stage.     

A standard set of polymorphic microsatellites for threatened mountain ungulates (Caprini,Artiodactyla)

Nearly 70% of the world's mountain ungulate taxa are endangered. The availability of a standard set of DNA markers for forensic and molecular ecology studies would help to establish conservation programs and detect poaching activities of these endangered taxa. We tested 60 published microsatellite primer pairs from bovids (cattle, sheep and goat) on 49 individuals from 11 taxa including six wild goat‐like species (Capra spp.), three divergent wild sheep (Ovis spp.), and two chamois (Rupicapra spp.) species. Approximately 30 microsatellites amplified a microsatellite‐like PCR product in all three genera, and with the exception of ILST097, nearly all the loci were polymorphic within most of the 11 species.