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.     

Genetic variation and differentiation of wood mice from the genus <Emphasis Type="Italic">Sylvaemus</Emphasis> inferred from sequencing of the cytochrome oxidase subunit 1 gene fragment

To ascertain intra- and interspecific differentiation patterns of some Sylvaemus wood mice species (S. uralensis, S. sylvaticus, S. ponticus, S. flavicollis, and S. fulvipectus), sequence variation of the fragment (654 bp) of the mitochondrial cytochrome oxidase subunit I gene (COI) was analyzed and the data obtained using several molecular genetic markers were compared. Distinct isolation of all Sylvaemus species (including closely related allopatric S. flavicollis and S. ponticus), as well as of the European and Asian races of pygmy wood mouse S. uralensis at the COI gene was demonstrated. However, genetic differences of the Sylvaemus species were 1.5 times and more higher than the distance (D) between the races of S. uralensis. This finding provides no ample grounds to treat them as the independent species. The only examined specimen of Pamir-Alay subspecies S. uralensis pallipes showed closest relatedness to to the Asian race, although was rather distant from it (D = 0.038). No reliable isolation of the eastern European and southern European chromosomal forms, representing the European race of S. uralensis, as well as of their presumptive hybrids from the out-skirts of the city of Sal’sk, Rostov region, at the COI gene was revealed. A hybrid origin of the population of pygmy wood mouse from the outskirts of the Talapker railway station, Novovarshavsky district, Omsk region, was confirmed (in preliminary studies, based on karyotypic characters, these specimens were diagnosed as distant hybrids of the eastern European chromosomal form and the Asian race). In yellow-necked wood mouse S. flavicollis from the territory of Russia and Ukraine, weak differentiation into northern and southern lineages (with mean genetic distance between them of 0.020) was observed. Considerably different relative genetic distances between the races of S. uralensis and the S. flavicollis-S. ponticus species pair, inferred from the mitochondrial cytochrome oxidase and cytochrome b genes data, indicated that the rates of evolution of the mitochondrial genome regions could be very dissimilar. It is suggested that transformations of the cytochrome b gene, or at least its part, were irregular in time and/or in different phyletic lineages (i.e., accelerated upon the formation of pygmy wood mouse races, and delayed upon the establishment of S. flavicollis and S. ponticus).     

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.     

Phenetic relationships among four Apodemus species (Rodentia, Muridae) inferred from skull variation

Phenetic relationships among four Apodemus species (A. agrarius, A. epimelas, A. flavicollis and A. sylvaticus) inferred from skull (mandible and cranium) variation were explored using landmark-based geometric morphometrics. Analysis of size variation revealed that mandibles and crania of A. epimelas were the largest, followed by those of A. flavicollis, while A. agrarius and A. sylvaticus had the smallest ones. Phenetic relationships inferred from mandible shape variation better reflected phylogenetic relationships among the analyzed Apodemus species than those inferred from cranial differences. Concerning cranial shape variation, the most differentiated species was A. epimelas, whose ecology clearly differs from the other three species. Thus, differentiation of the mandible provided a pattern fully concordant with the phylogeny, while the cranium differentiation was in agreement with ecology expectations. The most evident shape changes of mandible and cranium involved the angular process and facial region, respectively. We also found that allometry had a significant influence on shape variation and that size-dependent shape variation differed among the analyzed species. Moreover, mandible and cranium are differently influenced by allometric changes. Different phenetic relationships inferred from mandible and cranium shape variation imply that phylogeny, ecology, together with factors related to size differences are all involved in the observed morphological divergence among the analyzed Apodemus species.     

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.     

Comparative FISH analysis of C-positive regions of chromosomes of wood mice (Rodentia,Muridae, <Emphasis Type="Italic">Sylvaemus</Emphasis>)

The homology of DNA of C-positive centromeric regions of chromosomes in wood mice of the genus Sylvaemus (S. uralensis, S. fulvipectus, S. sylvaticus, S. flavicollis, and S. ponticus) was estimated for the first time. DNA probes were generated by microdissection from the centromeric regions of individual autosomes of each species, and their fluorescence in situ hybridization (FISH) with metaphase chromosomes of representatives of all studied wood mouse species was carried out. Unlike in the chromosomal forms and races of S. uralensis, changes in the DNA composition of the chromosomal centromeric regions in the wood mouse species of the genus Sylvaemus (including closely related S. flavicollis and S. ponticus) are both quantitative and qualitative. The patterns of FISH signals after in situ hybridization of the microdissection DNA probes with chromosomes of the species involved in the study demonstrate significant differences between C-positive regions of wood mouse chromosomes in the copy number and the level of homology of repetitive sequences as well as in the localization of homologous repetitive sequences. It was shown that C-positive regions of wood mouse chromosomes can contain both homologous and distinct sets of repetitive sequences. Regions enriched with homologous repeats were detected either directly in C-positive regions of individual chromosomes or only on the short arms of acrocentrics, or at the boundary of C-positive and C-negative regions.     

A Biogeographic View of Apodemus in Asia and Europe Inferred From Nuclear and Mitochondrial Gene Sequences

Sequences of the mitochondrial cyt b gene and nuclear IRBP, RAGI, I7, and vWF genes were used to assess the evolutionary history of major lineages of Apodemus, in particular to better understand dispersal between Asia and Europe. Our data show eight extant lineages of Late Tertiary origin: Apodemus agrarius, A. semotus, A. peninsulae, A. speciosus, A. argenteus, A. gurkha, A. mystacinus, and A. sylvaticus. Monophyly of two European lineages (A. mystacinus and A. sylvaticus) and four Asian lineages (A. agrarius, A. semotus, A. peninsulae, and A. speciosus) was confirmed with high bootstrap support. Together with literature data, the available molecular data depict three crucial evolutionary events: (1) initial wide dispersal and subsequent radiation around 6 million years ago, (2) region-specific radiations in Europe and southern China around 2 million years ago, and (3) westward dispersal of A. agrarius to Europe in the Late Quaternary.     

A Phylogenetic View on Species Radiation in Apodemus Inferred from Variation of Nuclear and Mitochondrial Genes

Species of field mice (genus Apodemus) are the most common rodents inhabiting woodlands and forests of the Palaearctic region. We examined the cytochrome b (cyt b) gene in mitochondrial DNA (1140 bp) and the interphotoreceptor retinoid binding protein (IRBP) gene in nuclear DNA (1152 bp) in nine species of Apodemus. Based on the genetic variation, the nine species were grouped into four lineages: (1) Agrarius group (A. agrarius, A. peninsulae, A. semotus, and A. speciosus), (2) Argenteus group (A. argenteus), (3) Gurkha group (A. gurkha), and (4) Sylvaticus group (A. alpicola, A. flavicollis, and A. sylvaticus). It was shown that these four lineages diverged within a short period of evolutionary time, suggestive of a radiation event. Soon after the radiation, the Agrarius group was likely to have differentiated again into the species lineages simultaneously. In contrast, the European clade, the Sylvaticus group, radiated rather recently. The relative ratio of the extent of sequence divergence among the four main lineages to that among the members of the subfamily Murinae (including Mus and Rattus) was calculated to be 72.4% in the cyt b gene with transversional substitutions, and 58.5% in the IRBP gene with all substitutions, using the Kimura two-parameter method. The value for the three European lineages was 27.6% in the cyt b gene and 12.3% in the IRBP gene. These results may have a correlation with the notion that deciduous broadleaf forests remained in Central East Asia through the late Tertiary to the present, while those in Europe to a large extent had disappeared by the Pliocene.     

Behavior of sex chromosomes at early meiosis stages in three wood mice species of the genus <Emphasis Type="Italic">Apodemus</Emphasis> (Rodentia,Muridae)

The prophase of the first meiotic division was studied in field mice of the species Apodemus (Sylvaemus) flavicollis, A. (S.) ponticus, and A. (S.) uralensis by light and electron microscopy. The karyotypes of three species were described on the base of electron microscopy of synaptonemal complexes in spermatocytes I. The axial elements of the sex chromosomes at early-middle pachytene synapse along the major portion of the Y axis; at late pachytene-early diplotene, the synapsis region shrinks; and at diakinesis-metaphase I, X and Y chromosomes associate end-to-end in all species studied. The behavior of sex chromosomes in the synapsis in the species studied was quite uniform. The results are discussed in the context of earlier data on the behavior of sex chromosomes in various rodent species in meiosis prophase I and their banding.     

Molecular Phylogeny of the Chipmunk Genus Tamias Based on the Mitochondrial Cytochrome Oxidase Subunit II Gene

Complete sequences of the mitochondrial cytochrome oxidase subunit II gene were used to construct a phylogeny for 21 of the 25 currently recognized chipmunk species. Phylogenetic analyses indicate that T. striatus (subgenus Tamias, eastern United States) and T. sibiricus (subgenus Eutamias, Asia) are distantly related to the other species (subgenus Neotamias), which constitute a western North American radiation. We discuss and compare our molecular phylogeny to previous taxonomies and present a suggested classification of the species groups for the subgenus Neotamias.     

A Three-Gene Dinoflagellate Phylogeny Suggests Monophyly of Prorocentrales and a Basal Position for <Emphasis Type="Italic">Amphidinium</Emphasis> and <Emphasis Type="Italic">Heterocapsa</Emphasis>

Many outstanding questions about dinoflagellate evolution can potentially be resolved by establishing a robust phylogeny. To do this, we generated a data set of mitochondrial cytochrome b (cob) and mitochondrial cytochrome c oxidase 1 (cox1) from a broad range of dinoflagellates. Maximum likelihood, maximum parsimony, and Bayesian methods were used to infer phylogenies from these genes separately and as a concatenated alignment with and without small subunit (SSU) rDNA sequences. These trees were largely congruent in topology with previously published phylogenies but revealed several unexpected results. Prorocentrum benthic and planktonic species previously placed in different clusters formed a monophyletic group in all trees, suggesting that the Prorocentrales is a monophyletic group. More strikingly, our analyses placed Amphidinium and Heterocapsa as early splits among dinoflagellates that diverged after the emergence of O. marina. This affiliation received strong bootstrap support, but these lineages exhibited relatively long branches. The approximately unbiased (AU-) test was used to assess this result using a three-gene (cob + cox1 + SSU rDNA) DNA data set and the inferred tree. This analysis showed that forcing Amphidinium or Heterocapsa to relatively more derived positions in the phylogeny resulted in significantly lower likelihood scores, consistent with the phylogenies. The position of these lineages needs to be further verified. Reviewing Editor: Dr. Martin Kreitman     

A mitochondrial DNA based phylogeny of weakfish species of the Cynoscion group (Pisces: Sciaenidae)

We infer the phylogeny of fishes in the New World Cynoscion group (Cynoscion, Isopisthus, Macrodon, Atractoscion, Plagioscion) using 1603 bp of DNA sequence data from three mitochondrial genes. With the exception of Plagioscion, whose position was ambiguous, the Cynoscion group is monophyletic. However, several genera examined are not monophyletic. Atlantic and Pacific species of Cynoscion are interspersed in the tree and geminate species pairs are identified. Intergeneric relationships in the group are clarified. Our analysis is the first comprehensive phylogeny for the Cynoscion group based on molecular data and provides a baseline for future comparative studies of this important group.     

Influence of age, sex, and sexual activity on trace element levels and antioxidant enzyme activities in field mice (Apodemus sylvaticus and Mus spretus)

The influence of age, gender and sexual activity on both hepatic levels of some trace elements (Fe, Cu, Zn, Mn, Se) and the activities of glutathione-S-transferase (GST) and superoxide dismutase (SOD) was investigated in Wood mice (Apodemus sylvaticus) and Algerian mice (Mus spretus). Animals were taken from a riverside community of an unpolluted area of central Portugal. Adult A. sylvaticus presented the highest hepatic mean concentrations of Cu and Mn, whereas adult M. spretus had the highest Fe concentration in the liver. Moreover, an influence of age on the contents of Fe, Zn, and Mn has been observed in A. sylvaticus, whereas in M. spretus an influence of gender and sexual activity was only detected on Zn levels. In contrast, enzyme activities were not influenced by the studied variables, despite a tendency for an increase in SOD activity in sexually active M. spretus. GST activity was species dependent, whereas SOD activity was similar between species. These findings were analyzed regarding the relationship of both essential trace elements and the two antioxidant enzymes with physiological and metabolic pathways related to life cycles in the two species of mice. Results enhanced the understanding of A. sylvaticus and M. spretus as biological models, allowing their future use as bioindicators of environmental toxicity.     

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.     

Evolution and phylogenetic relationships within Porinaceae (Ostropomycetidae), focusing on foliicolous species

A phylogeny of the lichen family Porinaceae using mitochondrial SSU rDNA sequences is presented, with special focus on foliicolous taxa. Fifty specimens of 28 mostly tropical species, representing eight species groups of Porina as well as the genus Trichothelium, were analysed together with species of other members of Ostropomycetidae, and using Agyriaceae as outgroup. We performed the phylogenetic analyses with a Bayesian approach and under the criterion of maximum parsimony. Four main clades can be distinguished: the P. nitidula-group s. lat. (including Trichothelium, P. papillifera and P. rubescens), the Porina epiphylla-group s. lat. (including the P. radiata-, the P. nucula-, the P. imitatrix- and the P. epiphylla-group s. str.) and two clades of the P. rufula-group. The genus Porina as understood by all recent concepts is paraphyletic, and Trichothelium is nested within the Porina nitidula-group. The non-setose P. repanda forms a monophyletic clade with Trichothelium. The tree does not support a monophyletic origin of substrate preferences or photobiont selection. Species-specific associations with morphologically different trentepohlioid photobionts mapped on the tree suggest that closely related mycobiont species switch between different types of algae.     

Identification of black muntjac (Muntiacus crinifrons) in Tibet, China, by cytochrome b analysis

Full cytochrome b gene sequence of mtDNA was used to identify and analyze four skin samples collected from Tibet, China in this research. By searching for highly similar sequences (megablast) on NCBI, we found all four samples have the highest similarities with the published sequence: AY239042 of the black muntjac (Muntiacus crinifrons). By comparing our sequences to those available on GenBank, all four samples were identified as the black muntjac (Muntiacus crinifrons) by high sequence similarity. We therefore record two new localities for the black muntjac and it is a new distribution in Tibet and hope this study will not only promote more advanced researches on the evolution and phylogeny about this species, but also enhance the conservation work for this species and local biodiversity.     

Molecular Systematics of Chipmunks (<Emphasis Type="Italic">Neotamias</Emphasis>) Inferred by Mitochondrial Control Region Sequences

Western chipmunks (Neotamias) exhibit a complex geographical distribution and can vary by minute morphological differences. This has lead to confusion around the taxonomy and evolutionary history of this group. The main focus of this molecular study was to infer taxonomic relationships within Neotamias, especially at the tips of the tree. Sequences of the complete control region for 16 species of chipmunks (Tamias, Neotamias) were analyzed independently and in combination with previously published sequences of two mitochondrial genes, cytochrome b and cytochrome oxidase II. The control region data set corroborated the findings of Piaggio and Spicer (2001) in finding five discrete clades, while also providing stronger bootstrap support for most terminal branches. Analysis of individual mitochondrial genes revealed that not all genes have the same phylogenetic signal and when analyzed in combination, this incongruence amongst genes is resolved.     

Phylogenetic analysis of Sesuvioideae (Aizoaceae) inferred from nrDNA internal transcribed spacer (ITS) sequences and morphological data

The phylogeny of the four genera of Aizoaceae subfamily Sesuvioideae (Sesuvium, Cypselea, Trianthema and Zaleya) is elucidated employing internal transcribed spacer (ITS) sequences and 23 morphological characters. Phylogenetic analysis based on ITS sequences and a combined molecular-morphological analysis provide largely congruent results. The monophyly of Sesuvioideae and its close relationship to Aizooideae s.l. and Mesembryanthemoideae is confirmed. Zaleya is placed within Trianthema and Cypselea within Sesuvium by ITS analysis, but in the combined analysis, Zaleya forms an unresolved polytomy with the two Trianthema clades and Cypselea, as well as two Sesuvium species, remain unresolved. Sesuvium sesuvioides and S. hydaspicum, previously treated as synonyms, are closely related, but molecular data do not support conspecifity. The Trianthema triquetra complex needs further intensive study because the African T. triquetra sample is closer to the NE African-Arabian T. sheilae than to Australian samples of T. triquetra. The close relationship of four species of Trianthema (T. patellitecta, T. rhynchocalyptra, T. megasperma, and T. pilosa) from Australia based on molecular data is supported morphologically by the exclusive possession of a well-developed indumentum in these taxa.     

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.