Discrimination of the sibling species Apodemus flavicollis and A. sylvaticus (Rodentia,Muridae)

Karyotyping and several molecular methods have allowed successful identification of two morphologically similar wide-ranging Western Palearctic species, the yellow-necked field mouse Apodemus flavicollis (Melchior, 1934) and the long-tailed wood mouse A. sylvaticus (Linnaeus, 1758), but reliable species diagnosis on the basis of morphometric characters is particularly problematic. Although they are easily morphologically distinguishable in Central and Northern Europe, this is not the case in southern parts of their distribution areas. Despite that, we have successfully discriminated A. flavicollis and A. sylvaticus from Serbia (Southern Europe) using geometric and traditional morphometric methods on a data set for ventral crania of specimens previously genotyped by the Inter Simple Sequence Repeat-PCR (ISSR-PCR). Discrimination power of applied approaches was more or less similar. The majority of our results were consistent with those obtained for specimens collected across the Czech Republic (Central Europe). Morphological differences observed herein, as well as those already reported between A. flavicollis and A. sylvaticus from the central and northern parts of their distribution areas, could be the outcome of their biology, i.e. ecological discrepancies, different assumed evolutionary scenarios considering biogeography, phylogeny, history and ontogeny.     

A removal experiment with sympatric populations of Apodemus sylvaticus (L.) and A. flavicollis (Melchior) (Rodentia: Muridae)

Summary This study examines the role of competition in the relationship between sympatric populations of Apodemus sylvaticus and A. flavicollis at Woodchester Park, Gloucestershire, England. Three grids were trapped for twelve months to monitor changes in population size, survival, reproduction, body weight, spatial segregation and microhabitat associations. The A. flavicollis was removed from one grid, A. sylvaticus from another while the rodent community on the third was left intact as a control. Subsequent changes in ecological parameters were observed over a further period of 7 months. Isolated populations of both species entered reproductive condition earlier than on the control grid. This resulted in a rapid increase of the A. flavicollis population but the dynamics of A. sylvaticus were unaffected. In the absence of their congener both species made greater use of areas of woodland once frequented by the absent species. However specific microhabitat associations observed in two species communities remained after experimental manipulation. It is concluded that although A. sylvaticus and A. flavicollis do compete for space, this is a comparatively weak interaction which does not lead to habitat exclusion. Competition is probably ameliorated by intrinsic differences in the biology of these Apodemus species.     

A chromosomal study on Greek populations of the genusApodemus (Rodentia, Murinae) reveals new data on B chromosome distribution

During this study, 94 specimens (51 males, 43 females) of the wood mouseApodemus sylvaticus (Linnaeus, 1758), the yellow-necked mouseA. flavicollis (Melchior, 1834) and the western broad-toothed mouseA. epimelas (Nehring, 1902) from 20 localities of Greece were karyologically examined. The first two species were found to be widely distributed and their otherwise very similar karyotype (2n=48, FN=48) could be clearly distinguished, based on C-banding pattern. The third species appeared to have a more limited geographical distribution and its karyotype was distinguished from that of the previous two species, since it contained two pairs of small metacentric autosomes (2n=48, FN=52). The chromosomal study further revealed that nine individuals ofA. flavicollis possessed supernumerary B chromosomes (2n=49–50, FN=49–50). Meiotic chromosome preparations revealed that in contrast to sex chromosomes and autosomes, B chromosomes do not participate in bivalent formation. On the other hand, no supernumerary chromosomes were found in the studiedA. sylvaticus andA. epimelas material.     

Differentiation of restriction sites in ribosomal DNA in the genusApodemus

Southern blot analysis of ribosomal DNA (rDNA) from seven species ofApodemus was carried out in order to examine the genetic relationships between the species. Analysis of heterogeneity in rDNA spacers inA. sylvaticus, A. flavicollis, A. semotus, A. agrarius, A. argenteus, A. speciosus, andA. peninsulae, using 13 different restriction enzymes and cloned mouse rDNA probes, revealed that the families of rDNA in these species can be characterized by restriction maps which show the major constituents of rDNA repeating units (repetypes). Based on differences in the arrangement of restriction sites, sequence divergence among the different major repetypes was estimated. Among the seven species ofApodemus examined, the major repetypes ofA. flavicollis andA. sylvaticus were the most closely related, having only 1.0% sequence divergence. These repetypes and those of the remaining five species differ substantially from one another, with 4.3–8.5% divergence.This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.     

Intra- and interspecific interactions of Apodemus sylvaticus (L.) and A. flavicollis (Melchoir) under laboratory conditions

Miller (1967) has indicated that the coexistence of Apodemus sylvaticus and A. flavicollis may be possible if A. flavicollis, a species with a more restricted niche, is competitively superior in interspecific encounters. Intra- and interspecific behaviour of male and female A. sylvaticus and A. flavicollis was examined in dyadic encounters in a small arena. Interactions between conspecifics indicate that intraspecific behaviour in A. sylvaticus and A. flavicollis is similar but, in interspecific interactions there were more submissive and agonistic acts, and less introductory and amicable behaviour. A. flavicollis was dominant to A. sylvaticus in 63 out of 70 encounters and A. sylvaticus dominant in only one. This superiority may contribute to the persistence of small, localized populations of A. flavicollis despite the presence of A. sylvaticus.     

Comparative cytogenetic analysis of the “Sylvaemus” group of Apodemus (Rodentia, Muridae): A. sylvaticus from Sicily and A. flavicollis from the central Apennines

Chromosome mapping of three common markers, ie major and 5S rRNA genes (rDNA) and telomeric repeats, and conventional chromosome bandings were applied to two sibling species, Apodemus sylvaticus Linnaeus, 1758 and A. flavicollis Melchior, 1834, to further investigate intra- and interspecific karyological differentiation in the genus Apodemus. A slight variation of the rDNA-patterns was detected between the two Apodemus species. In both of them, the major NORs were located on autosome pairs 8, 11, 12 and 22, while the two other rDNA sites detected on chromosomes 7 and 21 were variable in, respectively, A. sylvaticus and A. flavicollis. Several tiny rDNA sites were present on the sex chromosomes in both species, but their incidence was lower in A. flavicollis. Single 5S rDNA chromosomal sites were conserved on chromosome pair 20. No interstitial sites of telomeric repeats were present in either species. In the Sicilian population of A. sylvaticus, the constitutive heterochromatin pattern corresponded to the “sylvaticus-E1” cytotype, while A. flavicollis had a species-specific pattern restricted to centromeres of all chromosomes. The results are discussed in relation to cytogenetic data available for the genus, with emphasis on the Sylvaemus group/subgenus.     

A comparative study of the heterochromatin of Apodemus sylvaticus and Apodemus flavicollis

The two closely related species Apodemus sylvaticus and Apodemus flavicollis (Muridae) differ in the distribution of their heterochromatin. Two major repetitive sequences known to occur in both species were isolated from A. flavicollis after digestion of total nuclear DNA with the restriction enzymes HindIII and EcoRI respectively and characterized in both species by filter hybridisation and in situ hybridisation to metaphase chromosomes. The EcoRI clone detects a dispersed repetitive sequence family in the genome of both species. Southern blot hybridisation with the HindIII satellite DNA probe reveals major similarities and minor differences in the two species. In situ hybridisation with the HindIII probe labels all chromosomes of A. flavicollis exclusively in the centromeric heterochromatin, whereas in A. sylvaticus several autosomes are also labelled distally. The labelling patterns correspond to the distribution of heterochromatin in the two species. It is concluded that the additional distal heterochromatin of A. sylvaticus contains similar sequences to those of the centromeric heterochromatin of both species. The distal heterochromatin in A. sylvaticus most likely evolved by transposition and amplification of centromeric satellite DNA elements, after the separation of the two species.     

Comparative study of the phylogenetic structure in six Apodemus species (Mammalia,Rodentia) inferred from ISSR-PCR data

The utility of the Inter Simple Sequence Repeat-Polymerase Chain Reaction (ISSR-PCR) was explored in order to determine genetic variation in six species of the genus Apodemus (A. flavicollis, A. sylvaticus, A. uralensis, A. agrarius, A. mystacinus and A. epimelas) at the individual level, population level, in separate geographic samples and in the species as a whole. Six optimized primers produced highly reproducible and polymorphic DNA markers with 98.3% polymorphic bands on a total sample of 91 individuals from 32 localities in Europe and Asia. Moreover, each primer allowed for an exact diagnosis of each of the six Apodemus species and thus provides a simple and reliable tool for the hitherto problematic discrimination of species from the subgenus Sylvaemus. Genetic distances between species ranged from 0.079, among the closely related A. flavicollis and A. sylvaticus, to 0.203 between A. mystacinus and A. agrarius. A. flavicollis, A. uralensis and A. sylvaticus display a strong population substructure. The range of genetic distances among geographic samples within last two species reaches the values obtained for closely related species. ISSR markers proved to be a simple and reliable tool for species diagnosis, as well as for estimating genetic diversity below the species level and for closely related species, but they showed questionable reliability for larger genetic distances.     

A novel approach to field identification of cryptic Apodemus wood mice: calls differ more than morphology

Field identification of European wood mice Apodemus spp. is challenging due to their morphological resemblance and frequent sympatry. We developed discriminant functions based on body mass and acoustic variables of distress calls to identify three cryptic species of wood mice (Apodemus alpicola, Apodemus flavicollis and Apodemus sylvaticus) in Italy. We achieved an overall correct classification rate of 86–98%; the best results (100% correct classification) were obtained for Apodemus sylvaticus calls. This minimally invasive, effective and low‐cost method highlights the potential role of bioacoustics as a powerful tool for field discrimination of cryptic species of terrestrial mammals.     

A comparative study of mitotic and meiotic chromosomes of the Montandon's newt,Triturus montandoni (Urodela: Salamandridae) from Poland and Rumania

A karyological analysis was carried out on two populations ofTriturus montandoni, one from Poland and another from Rumania. For both samples, morphometric characteristics and C-banding pattern of mitotic chromosomes are provided. Only slight differences between specimens from two geographic localities were found. Data on chiasma frequency and distribution are presented for male meiosis. No sex-related heteromorphism was found and for none of the chromosomal arms was a consistent absence of chiasmata recorded. There was a relatively high proportion of spermatocyte metaphases I with chiasmata on both arms of all chromosomes in all specimens studied. It is concluded that there are no well-defined sex chromosomes in the chromosomal complement of the maleT. montandoni. The findings are compared with previous studies on chromosome morphology, C-banding pattern, and meiosis in closely related species,T. vulgaris andT. helveticus.     

No recent temporal changes in body size of three Danish rodents

We used museum collections to study temporal trends of possible changes in skull size, body mass and body length in three species of rodents in Denmark. Skulls of adult Microtus agrestis, Apodemus flavicollis and Apodemus sylvaticus, collected between 1895 and 2004, 1847 and 2002, and 1895 and 2002, respectively, were measured and data on body mass and length were taken from the museum registers. Principal component (PC) analysis was used to combine data of the four skull measurements taken. We tested the relationship of sex, latitude, longitude, month and year of collection to PC1 by a General Linear Model (GLM). PC1, body length and body mass of M. agrestis significantly increased from west to east. In addition, PC1, body mass and body length of M. agrestis declined from summer (August) through autumn and winter to spring (March), probably due to the decline in food availability towards winter. None of the other factors examined (sex, latitude and year) were significantly related to body size. PC1 of A. flavicollis and A. sylvaticus was not significantly related to any of the environmental factors examined.     

Evolution of MHC-<Emphasis Type="Italic">DRB</Emphasis> class II polymorphism in the genus <Emphasis Type="Italic">Apodemus</Emphasis> and a comparison of <Emphasis Type="Italic">DRB</Emphasis> sequences within the family Muridae (Mammalia: Rodentia)

Allelic diversity at major histocompatibility complex (MHC) genes is thought to be maintained by balancing selection over long periods of time, even across multiple speciation events. Trans-species sharing of MHC alleles among genera has been supported by many studies on mammals and fish, but in rodents, the results are ambiguous. We investigated natural levels of MHC-DRB variability and evolutionary processes in the wood mouse (Apodemus sylvaticus) and the yellow-necked mouse (Apodemus flavicollis), which are common, sympatric murid rodents in European forests. Using single-strand conformation polymorphism analysis and DNA sequencing, 38 DRB exon 2 alleles were detected among 162 A. sylvaticus from nine different locations in Germany and Switzerland, and 15 DRB exon 2 alleles were detected among 60 A. flavicollis from three different locations in northern Germany. There was evidence for balancing selection in both species. Phylogenetic analysis, including additional murid taxa, showed that the DRB exon 2 sequences did not separate according to species, consistent with trans-species evolution of the MHC in these taxa.     

Molecular identification of three sympatric species of wood mice (Apodemus sylvaticus,A. flavicollis,A. alpicola) in western Europe (Muridae: Rodentia)

The woodmouse (Apodemus sylvaticus) and yellow‐necked fieldmouse (Apodemus flavicollis) are sympatric and even syntopic in many regions throughout their European range. Their field discrimination on the basis of external characters is a real challenge for many fields of research. The problem is even more complicated in the Alpine chain where they live sympatrically with a third similar species: A. alpicola. A rapid and simple method is proposed to discriminate the three species in processing field‐collected biopsies as well as ethanol‐preserved museum samples.     

Morphological analysis of threeInia (Cetacea: Iniidae) populations from Colombia and Bolivia

We present new morphological data on three Colombian and BolivianInia populations. Analysis of morphometric data strongly suggests thatInia are sexually dimorphic, with males smaller for several variables and more heterogeneous than females. Our morphological data supported that Colombian Amazon males had the greatest total body length as well as greatest fluke values (tip to tip) compared to the males from the Orinoco and Bolivia Rivers. There were also significant differences in females among populations. The two Colombian groups (Amazon and Orinoco basin) presented greater total body length than the Bolivian array, while distance from jaw tip to tip of dorsal fin was greater in the Orinoco basin females than in the other two populations. A Canonic Population Analysis showed that the confidence intervals of the Colombian Amazon and Orinoco males were partially superposed. However, the Bolivian male sample was remarkably differing from these two populations. Two long standing debates exist in the systematic literature onInia: (1) the number of extant species (one,Inia geoffrensis, or twoI. geoffrensis andI. boliviensis) and (2) the number of extant subspecies ofI. geoffrensis (I. geoffrensis geoffrensis, I. geoffrensis humboldtiana andI. geoffrensis boliviensis). Using recent collections of both molecular and morphometric data, we suggest the existence of two distinct species ofInia with no subspecific distinction amongI. geoffrensis populations.     

Tick infestation of small mammals in an English woodland

Tick infestations on small mammals were studied from April to November, 2010, in deciduous woodland in southern England in order to determine whether co‐infestations with tick stages occurred on small mammals, a key requirement for endemic transmission of tick‐borne encephalitis virus (TBEV). A total of 217 small mammals was trapped over 1,760 trap nights. Yellow‐necked mice (Apodemus flavicollis) made up the majority (52.5%) of animals, followed by wood mice (A. sylvaticus) 35.5% and bank voles (Myodes glareolus) 12%. A total of 970 ticks was collected from 169 infested animals; 96% of ticks were Ixodes ricinus and 3% I. trianguliceps. Over 98% of ticks were larval stages. Mean infestation intensities of I. ricinus were significantly higher on A. flavicollis (6.53 ± 0.67) than on A. sylvaticus (4.96 ± 0.92) and M. glareolus (3.25 ± 0.53). Infestations with I. ricinus were significantly higher in August than in any other month. Co‐infestations with I. ricinus nymphs and larvae were observed on six (3.6%) infested individuals, and fifteen small mammals (8.9%) supported I. ricinus – I. trianguliceps co‐infestations. This work contributes further to our understanding of European small mammal hosts that maintain tick populations and their associated pathogens, and indicates that co‐infestation of larvae and nymph ticks does occur in lowland UK. The possible implications for transmission of tick‐borne encephalitis virus between UK ticks and small mammals are discussed.     

Taxonomic separation ofKengyilia (Poaceae: Triticeae) in relation to nearest relatedRoegneria,Elymus, andAgropyron,based on some morphological characters

Based on 100 species representative of the four genera, we scored 290 herbarium specimens for a number of morphological characters. The data were subjected to canonical discriminant analysis using characters different from those in the identification key to these genera byBaum, Yen, andYang (1991). These characters collectively support the four groupsAgropyron, Kengyilia, Roegneria, andElymus as previously defined. The four groups are also supported by the linear discriminant function with an overall rate of 83% correct classification. Length of lemma awn was found to be an additional diagnostic character asAgropyron andKengyilia have lemma awns shorter than 5 mm, whereasRoegneria andElymus have longer lemma awns with very few exceptions. Length of glume awns is also a useful supplementary generic diagnostic.Agropyron andElymus have glume awns, whereas the majority of species ofKengyilia and more than half of the species ofRoegneria lack them. If a glume awn is present it is usually not longer than 1 mm.     

Revision of the species composition of the wood mice from the genus <Emphasis Type="Italic">Sylvaemus</Emphasis> from the territory of Rostov oblast using karyological,allozyme and molecular genetic analysis

Using karyological, allozyme, and molecular genetic analysis, habitation of the four Sylvaemus wood mice species, pygmy wood mouse (S. uralensis), wood mouse (S. sylvaticus), yellow-necked mouse (S. flavicollis), and yellow-bellied mouse (S. fulvipectus) in Rostov oblast was demonstrated. Sylvaemus uralensis was distributed nearly over the whole territory of the oblast; S. sylvaticus was found in the central and western parts of the oblast on the right bank area of Don River; S. flavicollis inhabited northern and central parts of Rostov oblast on the right bank area of Don River; S. fulvipectus was found in the southern parts of the oblast, in the left bank area of Don River. Using the chromosome C-banding technique, it was demonstrated that the pygmy wood mice living in Rostov oblast in the right bank areas of Manych River and Don River in its low course, belonged to the eastern European chromosomal form of S. uralensis. The pygmy wood mice from the outskirts of the town of Salsk, the left bank area of Manych River, were probably hybrids between eastern European and southern European chromosomal forms. Based on the mitochondrial DNA cytochrome b gene fragment sequencing and chromosome C-banding, it was suggested that the wood mice inhabiting Rostov oblast belonged to the southern lineage of S. sylvaticus, living on Apennine Peninsula, Balkan Peninsula, and nearly throughout Ukraine.     

Geometric craniometric analysis of sexual dimorphism and ontogenetic variation: A case study based on two geographically disparate species, Aethomys ineptus from southern Africa and Arvicanthis niloticus from Sudan (Rodentia: Muridae)

Non-geographic morphometric variation, particularly at the level of sexual dimorphism and ontogenetic (age-related) variation, has been documented in rodents, and useful for establishing whether to analyse sexes separately or together, and for selecting adult specimens for subsequent data recording and analysis. However, such studies have largely been based on traditional morphometric analyses of linear measurements that mainly focus on overall size, rather than shape-related morphometric variation. Unit-free, landmark/outline-based geometric morphometric analyses are considered to offer a more appropriate tool for assessing shape-related morphometric variation. In this study, we used geometric cranial morphometric analysis to assess the nature and extent of sexual dimorphism and age variation within the Tete veld rat, Aethomys ineptus (Thomas and Wroughton, 1908) from southern Africa and the African Nile rat, Arvicanthis niloticus (Desmarest, 1822) from Sudan. The results obtained were in turn compared with previously published results based on independent geometric and traditional cranial morphometric data from the same sampled populations examined in the present study. While our geometric morphometric results detected statistically significant sexual dimorphism in cranial shape within Ar. niloticus only, previously published results based on traditional morphometric data failed to detect significant sexual dimorphism within this species. However, similar to previously published traditional morphometric data, our geometric morphometric results detected statistically significant age-related variation in cranial shape and size within both Ae. ineptus and Ar. niloticus, with individuals of age classes 5 and 6 being considered to represent adult specimens. Our results highlight the importance of carefully evaluating both size- and shape-related non-geographic morphometric variation prior to the analysis of geographic variation and the delineation of species. Erroneous conclusions of non-geographic variation may have implications in the interpretation of geographic and evolutionary processes that may be responsible for morphological differences at both the inter- and intra-specific levels.     

Morphological review ofPelvetia andSilvetia (Fucaceae, Phaeophyta) with an emphasis on phylogenetic relationships

We compared the morphology of all four members ofPelvetia andSilvetia (Fucaceae, Phaeophyta), with an emphasis on phylogenetic relationships.Silvetia is segregated fromPelvetia because it has two, longitudinally divided eggs in the oogonium. In contrast, the eggs of the genusPelvetia are transversally divided. A cladistic analysis, based on 17 morphological features, shows thatPelvetia is closely related toHesperophycus andPelvetiopsis, as are three species ofSilvetia. We can infer from the cladistic tree and biogeographic information that some silvetian ancestor populations from the northern Pacific region likely evolved toS. babingtonii in northern Japan and then moved to Korea and California (USA), whereS. siliquosa andS. compressa, respectively, diverged. Our morphological study corroborates the DNA-based phytogeny and the ensuing taxonomy for the two genera. These results demonstrate the necessity for systematically revising the family Fucaceae to emphasize egg development, rather than egg number, in the oogonium, as a diagnostic character.     

Isozyme studies in the genusCheirolophus (Asteraceae:Cardueae-Centaureinae) in the Iberian Peninsula, North Africa and the Canary Islands

The phylogenetic and phenetic analysis of 109 RAPD polymorphisms inS. nebrodensis, a perennial and self-incompatible endemic of four mountain ranges in Spain, andS. viscosus, a self-compatible annual widespread in Europe, as well asS. lividus, S. sylvaticus andS. vulgaris revealed a sister group relationship between the first two species. This result contrasts sharply with the earlier hypothesis based on isozyme variation thatS. viscosus originated from within a paraphyleticS. nebrodensis and that the two species represent a progenitor-derivative pair. After considering possible reasons for the sister group relationship found, including the possibility of rooting artefacts, it is concluded that neither the RAPD data nor the isozyme data allow to draw safe conclusions about the mode of speciation and therefore the relative age of the two species. As a consequence, the limited genetic variation ofS. viscosus in comparison toS. nebrodensis as revealed by both the RAPD and the isozyme data may reflect its population history, geographical distribution, reproductive ecology, or mode of dispersal just as well as its recent origin from a paraphyleticS. nebrodensis. The result of this study calls for a critical reexamination of other taxon pairs postulated to have a progenitor-derivative relationship on the basis of isozyme evidence.