Phylogeography and postglacial recolonization of Europe by Rhinolophus hipposideros: evidence from multiple genetic markers

The demographic history of Rhinolophus hipposideros (lesser horseshoe bat) was reconstructed across its European, North African and Middle‐Eastern distribution prior to, during and following the most recent glaciations by generating and analysing a multimarker data set. This data set consisted of an X‐linked nuclear intron (Bgn; 543 bp), mitochondrial DNA (cytb‐tRNA‐control region; 1630 bp) and eight variable microsatellite loci for up to 373 individuals from 86 localities. Using this data set of diverse markers, it was possible to determine the species’ demography at three temporal stages. Nuclear intron data revealed early colonization into Europe from the east, which pre‐dates the Quaternary glaciations. The mtDNA data supported multiple glacial refugia across the Mediterranean, the largest of which were found in the Ibero‐Maghreb region and an eastern location (Anatolia/Middle East)–that were used by R. hipposideros during the most recent glacial cycles. Finally, microsatellites provided the most recent information on these species’ movements since the Last Glacial Maximum and suggested that lineages that had diverged into glacial refugia, such as in the Ibero‐Maghreb region, have remained isolated. These findings should be used to inform future conservation management strategies for R. hipposideros and show the power of using a multimarker data set for phylogeographic studies.     

Across the southern Andes on fin: glacial refugia,drainage reversals and a secondary contact zone revealed by the phylogeographical signal of Galaxias platei in Patagonia

We employed DNA sequence variation at two mitochondrial (control region, COI) regions from 212 individuals of Galaxias platei (Pisces, Galaxiidae) collected throughout Patagonia (25 lakes/rivers) to examine how Andean orogeny and the climatic cycles throughout the Quaternary affected the genetic diversity and phylogeography of this species. Phylogenetic analyses revealed four deep genealogical lineages which likely represent the initial division of G. platei into eastern and western lineages by Andean uplift, followed by further subdivision of each lineage into separate glacial refugia by repeated Pleistocene glacial cycles. West of the Andes, refugia were likely restricted to the northern region of Patagonia with small relicts in the south, whereas eastern refugia appear to have been much larger and widespread, consisting of separate northern and southern regions that collectively spanned most of Argentinean Patagonia. The retreat of glacial ice following the last glacial maximum allowed re‐colonization of central Chile from nonlocal refugia from the north and east, representing a region of secondary contact between all four glacial lineages. Northwestern glacial relicts likely followed pro‐glacial lakes into central Chilean Patagonia, whereas catastrophic changes in drainage direction (Atlantic → Pacific) for several eastern palaeolakes were the likely avenues for invasions from the east. These mechanisms, combined with evidence for recent, rapid and widespread population growth could explain the extensive contemporary distribution of G. platei throughout Patagonia.     

Taxonomic revision of the marbled skink (Cyclodina oliveri,Reptilia: Scincidae) species complex,with a description of a new species

Abstract

We have completed a taxonomic revision of the New Zealand marbled skink (Cyclodina oliveri) species complex. Morphological analyses and mitochondrial sequence data (ND2, ND4, Cytochrome b; Total 1933 bp) are used to describe a new taxon (commonly known as the “Mokohinau” skink) and redefine C. oliveri. The morphological and molecular data indicate that C. oliveri is distributed on the Poor Knights Islands, Mercury Islands and Aldermen Islands. The new species is restricted to the Mokohinau Islands, Hen and Chickens group, Little Barrier Island and Great Barrier Island. Our data demonstrate that there is no support for the separation of the Poor Knights Islands population of C. oliveri from those on the Mercury Islands and Aldermen Islands. The genetic data indicate that C. whitakeri is part of the C. oliveri species group. Divergence time estimates indicate that the C. oliveri species complex diverged during the late‐Miocene, with further divergences among island groups in C. oliveri including the origin of the new taxon during the late‐Pliocene and mid‐Pleistocene. We present a diagnostic key for Cyclodina.     

Geographic distribution of an extinct equid (Equus hydruntinus: Mammalia, Equidae) revealed by morphological and genetical analyses of fossils

Equus hydruntinus inhabited Europe and the Middle East for more than 300 000 years. For a long time, palaeontological data failed to place E. hydruntinus into the equid phylogenetic tree, confronted with the fact that it shares primitive Equus characters with both zebras and asses, and derived characters with asses and hemiones. However, the study of a recently discovered skull points to a relationship with hemiones. Extraction of DNA from ancient samples from Crimea (E. hydruntinus) and Iran (E. cf. hydruntinus) yielded 134-288 bp of the mtDNA control region and 143 bp of the cytochrome b gene. This DNA analysis supports the proximity of E. hydruntinus and Equus hemionus suggested by skull and limb bone analyses, and rejects proximity to either Equus burchelli or the asses suggested by tooth morphology. Dental morphology may thus be of poor taxonomical value if used alone for establishing equid phylogenetic relationships. Furthermore, the small genetic distance between E. cf. hydruntinus of Iran and the classical E. hydruntinus of Crimea suggests that both samples belong to the same species. Accordingly, the geographic range of E. hydruntinus -- until now believed to be restricted to Europe, Israel, and Turkey -- can be extended towards East as far as Iran.     

Brief communication: Ancient nuclear DNA and kinship analysis: the case of a medieval burial in San Esteban Church in Cuellar (Segovia, Central Spain)

The aim of this work was to investigate a very common situation in the archaeological and anthropological context: the study of a burial site containing several individuals, probably related genetically, using ancient DNA techniques. We used available ancient DNA and forensic protocols to obtain reliable results on archaeological material. The results also enabled molecular sex determination to be compared with osteological data. Specifically, a modified ancient DNA extraction method combined with the amplification of nuclear markers with the AmpFlSTR®MiniFiler? kit(Applied Biosystems) was used. Seven medieval individuals buried in four niches dated in the 15th Century at San Esteban Church in Cuellar (Segovia, Central Spain) were analyzed by the proposed method, and four of seven provided complete autosomal short tandem repeat (STRs) profiles. Kinship analyses comprising paternity and sibship relations were carried out with pedigree‐specific software used in forensic casework. A 99.98% paternity probability was established between two individuals, although lower percentages (68%) were obtained in other cases, and some hypothetical kinship relations were excluded. The overall results could eventually provide evidence for reconstructing the historical record. Am J Phys Anthropol, 2011. © 2010 Wiley‐Liss, Inc.     

A preliminary analysis of the DNA and diet of the extinct Beothuk: a systematic approach to ancient human DNA

We have used a systematic protocol for extracting, quantitating, sexing and validating ancient human mitochondrial and nuclear DNA of one male and one female Beothuk, a Native American population from Newfoundland, which became extinct approximately 180 years ago. They carried mtDNA haplotypes, which fall within haplogroups X and C, consistent with Northeastern Native populations today. In addition we have sexed the male using a novel-sexing assay and confirmed the authenticity of his Y chromosome with the presence of the Native American specific Y-QM3 single nucleotide polymorphism (SNP). This is the first ancient nuclear SNP typed from a Native population in the Americas. In addition, using the same teeth we conducted a stable isotopes analysis of collagen and dentine to show that both individuals relied on marine sources (fresh and salt water fish, seals) with no hierarchy seen between them, and that their water sources were pooled or stored water. Both mtDNA sequence data and Y SNP data hint at possible gene flow or a common ancestral population for both the Beothuk and the current day Mikmaq, but more importantly the data do not lend credence to the proposed idea that the Beothuk (specifically, Nonosabasut) were of admixed (European-Native American) descent. We also analyzed patterns of DNA damage in the clones of authentic mtDNA sequences; there is no tendency for DNA damage to occur preferentially at previously defined mutational hotspots, suggesting that such mutational hotspots are not hypervariable because they are more prone to damage.     

Mutagenesis by N-nitroso compounds: relationships to DNA adducts, DNA repair, and mutational efficiencies

The relationships between DNA alkylation, DNA repair and mutagenesis by N-nitroso compounds in Salmonella were examined. DNA adducts formed by treatment of the bacteria with N-nitroso compounds were monitored. Critical to the study was establishing which adducts led to mutations. Two methods were employed. In one, correlations in the dose-responses for adducts and mutagenesis were sought. For instance O⁶-methyl- and -ethyl-guanine, in contrast to other adducts, exhibited thresholds in their accumulation in Salmonella DNA, and mutagenesis at GC base pairs also exhibited the same threshold, suggesting a dependence of mutagenesis on the O⁶-alkyguanines. In the second method, mutagenesis induced by different mutagens with overlapping adduct spectra was compared. For example, EMS and ENU generate similar ratios of adenine adducts, but only ENU produces thymine adducts, and only ENU induced AT-GC and AT-CG base changes. These observations suggested that ethylthymines led to these mutations. Furthermore, it was found that these mutations were largely dependent on the presence of the plasmid, pKM101, indicating that error-prone repair activity contributes importantly in their processing to mutations. When DNA adducts by N-nitrosopyrrolidine were examined it was found that only one major adduct was detected in an excision-repair-deficient strain, and that this adduct was not present in a repair-proficient strain. Mutagenesis was also greatly reduced in the proficient strain, suggesting that mutagenesis was dependent on this adduct. From the relationships between premutagenic adducts levels adducts. This calculation assumed an average distribution of adducts and mutations and required knowledge of the target size and the types of mutations that could lead to phenotypic changes. For the unrepaired O⁶-methyl- and -ethyl-guanines, and the O-ethylthymines the mutational efficiencies were high (ca. 30–70%), but for the N-nitrosopyrrolidine adduct it was low (ca. 1%). Initial studies were carried out on the mutational specificities of two higher homologue N-nitroso compounds (the N-nitroso-N-propyl- and N-butyl-nitroguanidines) in uvrB/pKM101 strains. This class of nitroso compounds is known to form similar DNA adducts as ENU. Their specificities were similar to that of N-nitroso-N-ethylurea at a high dose except the fraction of mutations at AT base pairs was reduced. The fraction of GC-CG transversions was although low, increased. The mutational specificities of N-nitroso-N-methylurea and N-nitrosopyrrolidine were significantly different from the specificity of ENU as would be expected from their different adduct distributions.     

Phylogeography of the greater horseshoe bat, Rhinolophus ferrumequinum: contrasting results from mitochondrial and microsatellite data

Phylogeographical studies are typically based on haplotype data, occasionally on nuclear markers such as microsatellites, but rarely combine both. This is unfortunate because the use of markers with contrasting modes of inheritance and rates of evolution might provide a more accurate and comprehensive understanding of a species' history. Here we present a detailed study of the phylogeography of the greater horseshoe bat, Rhinolophus ferrumequinum , using 1098 bp of the mitochondrial ND2 gene from 45 localities from across its Palaearctic range to infer population history. In addition, we re-analysed a large microsatellite data set available for this species and compared the results of both markers to infer population relationships and the historical processes influencing them. We show that mtDNA, the most popular marker in phylogeography studies, yielded a misleading result, and would have led us to conclude erroneously that a single expansion had taken place in Europe. Only by combining the mitochondrial and microsatellite data sets are we able to reconstruct the species' history and show two colonization events in Europe, one before the Last Glacial Maximum (LGM) and one after it. Combining markers also revealed the importance of Asia Minor as an ancient refugium for this species and a source population for the expansion of the greater horseshoe bat into Europe before the LGM.     

Biogeography and evolution of European cave salamanders, Hydromantes (Urodela: Plethodontidae), inferred from mtDNA sequences

Aim To infer the phylogenetic relationships and biogeography of Hydromantes, with special emphasis on the European taxa. In particular, we aimed to test: (1) the monophyly of the European species and current views on their interrelationships; and (2) previously proposed timings of the separation of European and American Hydromantes, and of biogeographically important events within Europe. Location California and the Western Mediterranean Basin, specifically south‐east France, Italy, and the island of Sardinia. Methods Partial sequences of mitochondrial genes (cytochrome b and 12S rRNA) were obtained from 45 specimens of Hydromantes, including all European extant species and subspecies, and two species from California. In addition, a fragment of the mitochondrial 16S rRNA gene was amplified for 16 specimens. Data sets were aligned using Clustal X, and well‐supported phylogenetic trees were produced using maximum‐likelihood, Bayesian and maximum‐parsimony methods. Estimates of divergence times were obtained with the program r8s , the molecular clock being calibrated using the opening of the Strait of Gibraltar, the final event in the Messinian Salinity Crisis of 5.3 Ma. Results Separation between the American and European clades occurred approximately 13.5 Ma, most probably before or after westward dispersal across the Bering Land Bridge. In Europe, divergence started in the late Miocene, when Hydromantes (A.) genei separated from other members of the genus 9 Ma and colonized south‐west Sardinia. Movement between the European mainland and Sardinia, by a member of the subgenus Speleomantes, occurred in the Messinian Salinity Crisis, after the Mediterranean Basin desiccated almost completely 5.96 Ma. Subsequent widespread aridification fragmented the geographical ranges of Hydromantes, which live in cool and humid conditions, resulting in the origin of the six species in the subgenus Speleomantes. In contrast, a second period of diversification, in continental Europe 2–1.3 Ma, was probably caused by very cold interludes during the climatic oscillations that characterized the Pleistocene. Main conclusions The molecular clock used here indicates that the separation of Californian and European Hydromantes occurred more recently than previously believed, and the same is true of some subsequent phylogenetic divergences within Europe. Estimated dates for these divergence events are consistent with known geophysical and climatic events that could have caused or facilitated them.     

Genetic structure,phylogeography, and demography of Anadara tuberculosa (Bivalvia) from East Pacific as revealed by mtDNA: Implications to conservation

Wild populations of the pustulose ark, Anadara tuberculosa (Bivalvia), an emblematic species of the East Pacific mangrove ecosystem declined in South American countries (Colombia, Ecuador, and Peru) mainly due to overharvesting and habitat loss or degradation. Understanding the genetic aspects of geographic variations and population structure of A. tuberculosa, currently unknown, appears as a priority to fishery authorities in order to elaborate integrated and collaborative conservation policies for fishery management, aquaculture, and stock enhancement programs. We used mtDNA sequence data to investigate haplotype diversity, genetic structure, and demography of A. tuberculosa. Results indicate genetic homogeneity of populations distributed north and south of the equator, respectively. However, statistically significant differentiation emerged between northern and southern populations with pairwise ф_ST values ranging between 0.036 and 0.092. The oceanic current system acting in the area (Panama Current and Humboldt Current) might play a role in limiting the larval dispersal of the species, still poorly understood. Demography reconstruction supported recent population expansion, possibly started after last glacial maximum. Our results would suggest separate and independent management of populations north and south of the equator.     

Birds,Cattle, and Bracken Ferns: Bird Community Responses to a Neotropical Landscape Shaped by Cattle Grazing Activities

Large areas of tropical moist forests have been converted to cattle pastures, generating complex landscapes where different habitats are represented by small patches with an uneven spatial distribution. Here, we describe how bird communities respond to the different elements present in a livestock landscape that was originally dominated by tropical moist forest. We surveyed six habitats: open pastures, pastures with shrubs, early‐ and middle‐secondary forests, mature forest, and pastures invaded by bracken ferns (Pteridium aquilinum). Bird diversity was high in secondary and mature forests, and low in fern‐invaded sites and open pastures. Fern‐dominated sites had the lowest bird species richness, and trophic guild diversity of all habitats. Habitat structure affected both bird species richness and densities in similar ways. Tree species richness was the habitat attribute that had a bigger positive effect on bird species richness. Bird community structure varied among sampled habitats, separating habitats in two major groups (forests and pastures). Our data indicate that bracken fern‐invaded pastures were the worst habitat condition for avian communities. To increase bird diversity, we recommend to eliminate or manage bracken fern and to increase shrub and tree cover in open pastures to provide food resources and shelter for birds. Finally, we encourage the maintenance of secondary and mature forest remnants as a strategy to conserve resident birds within a landscape dominated by livestock activities.     

The Balkans and the colonization of Europe: the post‐glacial range expansion of the wild boar,Sus scrofa

Aim We focus on the biogeographical role of the Balkan Peninsula as a glacial refugium and source of northward post‐glacial dispersal for many European taxa. Specifically, we analysed the genetic structure and variation of wild boar (Sus scrofa) samples primarily from Greece, a region that has repeatedly served as a glacial refugium within the Balkan Peninsula. Location Continental Greece, the Aegean island of Samos and Bulgaria. Methods We analysed wild boar samples from 18 localities. Samples from common domestic breeds were also examined to take into account interactions between wild and domesticated animals. Phylogenetic analyses were carried out on a 637‐bp fragment of the mitochondrial DNA control region in 200 wild boar and 27 domestic pigs. The sequences were also compared with 791 Eurasian wild boar and domestic pig D‐loop sequences obtained from GenBank. Results Ninety‐four haplotypes were identified in the European wild boar data set, of which 68 were found in the Balkan samples and assigned to two previously described clades: the E1 European and Near Eastern clades. All of the continental samples clustered in the E1 clade and the samples from Samos fell into the Near Eastern clade, consistent with the island’s proximity to Asia Minor. Intriguingly, 62 novel haplotypes were identified and are found exclusively in the Balkans. Only six haplotypes were shared between wild boar and domestic pigs. Main conclusions Our data reveal numerous novel and geographically restricted haplotypes in wild boar populations, suggesting the presence of separate refugia in the Balkans. Our analyses support the hypothesis of a post‐glacial wild boar expansion consistent with the leading edge model, north and west from modern day Greece, and suggest little maternal introgression of Near Eastern and domestic haplotypes into wild Balkan populations.     

Tuberculosis in Dr Granville's mummy: a molecular re-examination of the earliest known Egyptian mummy to be scientifically examined and given a medical diagnosis

‘Dr Granville''s mummy’ was described to the Royal Society of London in 1825 and was the first ancient Egyptian mummy to be subjected to a scientific autopsy. The remains are those of a woman, Irtyersenu, aged about 50, from the necropolis of Thebes and dated to about 600 BC. Augustus Bozzi Granville (1783–1872), an eminent physician and obstetrician, described many organs still in situ and attributed the cause of death to a tumour of the ovary. However, subsequent histological investigations indicate that the tumour is a benign cystadenoma. Histology of the lungs demonstrated a potentially fatal pulmonary exudate and earlier studies attempted to associate this with particular disease conditions. Palaeopathology and ancient DNA analyses show that tuberculosis was widespread in ancient Egypt, so a systematic search for tuberculosis was made, using specific DNA and lipid biomarker analyses. Clear evidence for Mycobacterium tuberculosis complex DNA was obtained in lung tissue and gall bladder samples, based on nested PCR of the IS6110 locus. Lung and femurs were positive for specific M. tuberculosis complex cell-wall mycolic acids, demonstrated by high-performance liquid chromatography of pyrenebutyric acid–pentafluorobenzyl mycolates. Therefore, tuberculosis is likely to have been the major cause of death of Irtyersenu.     

Island Populations, Human Introductions and the Limitations of Genetic Analyses: the Case of the Sardinian Red Deer (Cervus elaphus corsicanus)

The Corsican red deer (Cervus elaphus corsicanus) is endemic to the Tyrrhenian islands of Corsica and Sardinia. It has been regarded as an introduced species and has allegedly been present on the islands since the beginning of the Neolithic culture some 8,000 years ago. In this review, we present the results of relevant genetic analyses and discuss their implications for the origin of C. e. corsicanus. Different genetic studies hypothesize that the most probable ancestral populations for Sardianian red deer were alternatively, the Near East, North Africa, or mainland Italy. These respective scenarios are evaluated and it is concluded that geneticists have not yet been unable to definitively solve the problem. However, a natural colonization of the Tyrrhenian islands from mainland Italy via the Tuscan archipelago is not only in accordance with palaeontological findings but also with at least some of the genetic data.     

Phylogeography of the mountain chickadee (Poecile gambeli): diversification, introgression, and expansion in response to Quaternary climate change

Since the late 1990s, molecular techniques have fuelled debate about the role of Pleistocene glacial cycles in structuring contemporary avian diversity in North America. The debate is still heated; however, there is widespread agreement that the Pleistocene glacial cycles forced the repeated contraction, fragmentation, and expansion of the North American biota. These demographic processes should leave genetic 'footprints' in modern descendants, suggesting that detailed population genetic studies of contemporary species provide the key to elucidating the impact of the late Quaternary (late Pleistocene-Holocene). We present an analysis of mitochondrial DNA (mtDNA) variation in the mountain chickadee (Poecile gambeli) in an attempt to examine the genetic evidence of the impact of the late Quaternary glacial cycles. Phylogenetic analyses reveal two strongly supported clades of P. gambeli: an Eastern Clade (Rocky Mountains and Great Basin) and a Western Clade (Sierra Nevada and Cascades). Post-glacial introgression is apparent between these two clades in the Mono Lake region of Central California. Within the Eastern Clade there is evidence of isolation-by-distance in the Rocky Mountain populations, and of limited gene flow into and around the Great Basin. Coalescent analysis of genetic variation in the Western Clade indicates that northern (Sierra Nevada/Cascades) and southern (Transverse/Peninsular Ranges) populations have been isolated and evolving independently for nearly 60,000 years.     

Fragmentation by weirs in a riverine system: A study of genetic variation in time and space among populations of European grayling (Thymallus thymallus) in a Danish river system

Human induced habitat alterations affect the genetic structure of many fish populations. Weirs in particular have caused fragmented populations previously connected by gene-flow. We studied the effects of weirs on the distribution of genetic variation within European grayling (Thymallus thymallus) populations from the Skjern River, Denmark. We compared microsatellite data from DNA extracted from historic scale samples collected 60 years ago with data from contemporary samples. Pairwise multilocus F _ST estimates between all contemporary population samples were significant as well as exact tests for population differentiation. Assignment tests of individuals to a set of baseline samples showed correct assignment to the population of origin of between 54 and 79%. Assignment of individuals from recent samples to the historic population samples showed highly variable results (3–83%) of correct assignment suggesting different population histories. Pairwise multilocus F _ST estimates were significantly correlated with the number of intervening weirs but not with waterway distance. A simulation procedure was used to estimate differences in relative population sizes, which indicated that the main river population was approximately three times larger than those of the tributaries. There were no signs of any loss of genetic variation for the river system as a whole during these 60 years. The results show that weirs can be an important factor for creating the current distribution patterns of genetic variation among grayling populations, most likely by only allowing passive downstream drift of fry and obstructing active upstream migration. This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular scatology: the use of molecular genetic analysis to assign species, sex and individual identity to seal faeces

Seals and commercial fisheries are potential competitors for fish and cephalopods. Research into the diet of British seal species has been based on conventional dietary analyses, but these methods often do not allow assignment of species identity to scat samples. We present a protocol for obtaining DNA from seal scat (faecal) samples which can be used in polymerase chain reactions to amplify both nuclear and mitochondrial DNA. This can provide a method of identifying the species, sex and individual identity of the seal, from a particular scat sample. Combined with conventional dietary analyses these techniques will allow us to assess sources of variation in seal diet composition.
Scat samples have been collected from intertidal haul-out sites around the inner Moray Firth, north-east Scotland. We have assessed methods to extract and purify faecal DNA, a combination of DNA from the individual seal, prey items, and gut bacteria, for use in PCR. Controls using faecal and blood samples from the same individual have enabled microsatellite primer sets from four pinniped species to be tested. Approximately 200 scat samples have been examined for species identity and individual matches. This study will provide essential information for the assessment of interactions between seals and commercial or recreational fisheries.     

DNA isolation by a rapid method from human blood samples: Effects of MgCl2, EDTA,storage time,and temperature on DNA yield and quality

The isolation of DNA from whole blood by a modified rapid method (RM) was tested using various detergents and buffer conditions. Extraction of DNA with either NP-40 or Triton X-100 gave a high yield of undegraded DNA in less than an hour. The concentration of magnesium ion in the buffers was critical to obtaining intact, high molecular weight (HMW) DNA. Greater than 10 mM MgCl₂ led to degradation. Addition of EDTA to the buffer inhibits this degradation. Preparation of DNA from blood stored at room temperature or incubated at 37°C for 24 hr resulted in the same amount and quality of DNA as from samples frozen at −70°C. DNA from blood samples that had undergone more than four freeze-thaw cycles was found to be partially degraded. The modified RM can be applied to extract DNA from as little as 10 μl of blood (340 ng of DNA) and from dried blood samples. DNA samples remained intact and undegraded for longer times when DNA was dissolved in higher concentrations of EDTA. This work was supported by grants from the Indiana Department of Mental Health and PHS RO1 AG10297.