Soil DNA metabarcoding and high-throughput sequencing as a forensic tool: considerations, potential limitations and recommendations.

Analysis of physical evidence is typically a deciding factor in forensic casework by establishing what transpired at a scene or who was involved. Forensic geoscience is an emerging multi-disciplinary science that can offer significant benefits to forensic investigations. Soil is a powerful, nearly 'ideal' contact trace evidence, as it is highly individualistic, easy to characterise, has a high transfer and retention probability, and is often overlooked in attempts to conceal evidence. However, many real-life cases encounter close proximity soil samples or soils with low inorganic content, which cannot be easily discriminated based on current physical and chemical analysis techniques. The capability to improve forensic soil discrimination, and identify key indicator taxa from soil using the organic fraction is currently lacking. The development of new DNA sequencing technologies offers the ability to generate detailed genetic profiles from soils and enhance current forensic soil analyses. Here, we discuss the use of DNA metabarcoding combined with high-throughput sequencing (HTS) technology to distinguish between soils from different locations in a forensic context. Specifically, we provide recommendations for best practice, outline the potential limitations encountered in a forensic context and describe the future directions required to integrate soil DNA analysis into casework.

Did postglacial sea-level changes initiate the evolutionary divergence of a Tasmanian endemic raptor from its mainland relative?

Populations on continental islands are often distinguishable from mainland conspecifics with respect to body size, appearance, behaviour or life history, and this is often congruent with genetic patterns. It is commonly assumed that such differences developed following the complete isolation of populations by sea-level rise following the Last Glacial Maximum (LGM). However, population divergence may predate the LGM, or marine dispersal and colonization of islands may have occurred more recently; in both cases, populations may have also diverged despite ongoing gene flow. Here, we test these alternative hypotheses for the divergence between wedge-tailed eagles from mainland Australia (Aquila audax audax) and the threatened Tasmanian subspecies (Aquila audax fleayi), based on variation at 20 microsatellite loci and mtDNA. Coalescent analyses indicate that population divergence appreciably postdates the severance of terrestrial habitat continuity and occurred without any subsequent gene flow. We infer a recent colonization of Tasmania by marine dispersal and cannot discount founder effects as the cause of differences in body size and life history. We call into question the general assumption of post-LGM marine transgression as the initiator of divergence of terrestrial lineages on continental islands and adjacent mainland, and highlight the range of alternative scenarios that should be considered.

Historical stocking data and 19th century DNA reveal human-induced changes to native diversity and distribution of cutthroat trout.

Many species are threatened with extinction and efforts are underway worldwide to restore imperilled species to their native ranges. Restoration requires knowledge of species' historical diversity and distribution. For some species, many populations were extirpated or individuals moved beyond their native range before native diversity and distribution were documented, resulting in a lack of accurate information for establishing restoration goals. Moreover, traditional taxonomic assessments often failed to accurately capture phylogenetic diversity. We illustrate a general approach for estimating regional native diversity and distribution for cutthroat trout in the Southern Rocky Mountains. We assembled a large archive of historical records documenting human-mediated change in the distribution of cutthroat trout (Oncorhynchus clarkii) and combined these data with phylogenetic analysis of 19th century samples from museums collected prior to trout stocking activities and contemporary DNA samples. Our study of the trout in the Southern Rocky Mountains uncovered six divergent lineages, two of which went extinct, probably in the early 20th century. A third lineage, previously declared extinct, was discovered surviving in a single stream outside of its native range. Comparison of the historical and modern distributions with stocking records revealed that the current distribution of trout largely reflects intensive stocking early in the late 19th and early 20th century from two phylogenetically and geographically distinct sources. Our documentation of recent extinctions, undescribed lineages, errors in taxonomy and dramatic range changes induced by human movement of fish underscores the importance of the historical record when developing and implementing conservation plans for threatened and endangered species.

Diversification patterns of pebble-mimic dragons are consistent with historical disruption of important habitat corridors in arid Australia.

The pebble-mimic dragon lineage of Tympanocryptis is widely distributed in the stony, or 'gibber', deserts of Australia but is noticeably absent from intersecting areas of sand deserts. Past fluctuations in the extent and configuration of sandy desert habitat barriers are likely to have been an import factor promoting genetic differentiation in this group. We sequenced a approximately 1400bp region of mitochondrial DNA and a approximately 1400bp nuclear gene (RAG-1) to investigate phylogeographic structuring of species of pebble-mimic dragons. Our topology indicates an early split in this lineage between eastern and western parts of the arid zone that probably dates to the mid-Miocene. This split corresponds directly with large expanses of contemporary sandy habitat in the form of Great Sandy and Great Victoria Deserts. Our data indicate that this biogeographic barrier established very early on in the development of the arid zone and has persisted to present. Additional genetic structuring in the absence of recognized barriers suggests that an expanded view of potential habitat barriers in the arid zone is required.

Genetic population structure and call variation in a passerine bird, the satin bowerbird, Ptilonorhynchus violaceus.

Geographic variation in vocalizations is widespread in passerine birds, but its origins and maintenance remain unclear. One hypothesis to explain this variation is that it is associated with geographic isolation among populations and therefore should follow a vicariant pattern similar to that typically found in neutral genetic markers. Alternatively, if environmental selection strongly influences vocalizations, then genetic divergence and vocal divergence may be disassociated. This study compared genetic divergence derived from 11 microsatellite markers with a metric of phenotypic divergence derived from male bower advertisement calls. Data were obtained from 16 populations throughout the entire distribution of the satin bowerbird, an Australian wet-forest-restricted passerine. There was no relationship between call divergence and genetic divergence, similar to most other studies on birds with learned vocalizations. Genetic divergence followed a vicariant model of evolution, with the differentiation of isolated populations and isolation-by-distance among continuous populations. Previous work on Ptilonorhynchus violaceus has shown that advertisement call structure is strongly influenced by the acoustic environment of different habitats. Divergence in vocalizations among genetically related populations in different habitats indicates that satin bowerbirds match their vocalizations to the environment in which they live, despite the homogenizing influence of gene flow. In combination with convergence of vocalizations among genetically divergent populations occurring in the same habitat, this shows the overriding importance that habitat-related selection can have on the establishment and maintenance of variation in vocalizations.

Using ancient and recent DNA to explore relationships of extinct and endangered Leiolopisma skinks (Reptilia: Scincidae) in the Mascarene islands.

Phylogenetic analysis, using 1455 bp of recent mtDNA (cytochrome b 714 bp, 12S rRNA 376 bp) and nuclear (c-mos 365 bp) sequence from 42 species and 33 genera of Scincidae, confirms Leiolopisma telfairii, now confined to Round island off Mauritius, is a member of the mainly Australasian Eugongylus group of the Lygosominae. Ancient mtDNA (cytochrome b 307 bp, 12S rRNA 376 bp) was also extracted from subfossils of two other Mascarene taxa that are now extinct: the giant L. mauritiana from Mauritius and Leiolopisma sp., known only from fragmentary remains from Réunion. Sequence divergences of 4.2-5.7% show that all three forms were distinct and form a clade. There is restricted evidence that L. mauritiana and L. sp. from Réunion were sister species. Monophyly and relationships suggest Leiolopisma arose from a single transmarine invasion of the oceanic Mascarene islands from Australasia, 5600-7000 km away. This origin is similar to that of Cryptoblepharus skinks and Nactus geckos in the archipelago but contrasts with Phelsuma day geckos, which appear to have arrived from Madagascar where Mascarene Cylindraspis tortoises may also have originated. Diversification of the known species of Leiolopisma occurred from about 2.3-3.4 Mya, probably beginning on Mauritius with later invasion of Réunion. The initial coloniser may have had a relatively large body-size, but L. mauritiana is likely to have become gigantic within the Mascarenes. Other relationships supported by this investigation include the following. Scincines: Pamelaescincus+Janetaescincus, and Androngo (Amphiglossus, Paracontias). Lygosomines: Sphenomorphus group--(Sphenomorphus, Lipinia (Ctenotus, Anomalopus (Eulamprus and Gnypetoscincus))): Egernia group--Egernia (Cyclodomorphus, Tiliqua); Eugongylus group--(Oligosoma, Bassiana. (Lampropholis (Niveoscincus, Carlia))).

Phylogeography of an east Australian wet-forest bird, the satin bowerbird (Ptilonorhynchus violaceus), derived from mtDNA, and its relationship to morphology.

Australian wet forests have undergone a contraction in range since the mid-Tertiary, resulting in a fragmented distribution along the east Australian coast incorporating several biogeographical barriers. Variation in mitochondrial DNA and morphology within the satin bowerbird was used to examine biogeographical structure throughout almost the entire geographical extent of these wet forest fragments. We used several genetic analysis techniques, nested clade and barrier analyses, that use patterns inherent in the data to describe the spatial structuring. We also examined the validity of the two previously described satin bowerbird subspecies that are separated by well-defined biogeographical barriers and tested existing hypotheses that propose divergence occurs within each subspecies across two other barriers, the Black Mountain corridor and the Hunter Valley. Our data showed that the two subspecies were genetically and morphologically divergent. The northern subspecies, found in the Wet Tropics region of Queensland, showed little divergence across the Black Mountain corridor, a barrier found to be significant in other Wet Tropics species. Biogeographical structure was found through southeastern Australia; three geographically isolated populations showed genetic differentiation, although minimal divergence was found across the proposed Hunter Valley barrier. A novel barrier was found separating inland and coastal populations in southern New South Wales. Little morphological divergence was observed within subspecies, bar a trend for birds to be larger in the more southerly parts of the species' range. The results from both novel and well-established genetic analyses were similar, providing greater confidence in the conclusions about spatial divergence and supporting the validity of these new techniques.

Reconstructing an island radiation using ancient and recent DNA: the extinct and living day geckos (Phelsuma) of the Mascarene islands.

Mitochondrial (12SrRNA and cyt b, 1086 bp) and nuclear (c-mos, 374 bp) DNA sequences were used to investigate relationships and biogeography of 24 living and extinct taxa of Phelsuma geckos. Monophyly of Phelsuma and sister relationship to the SW African Rhoptropella is corroborated. Phelsuma originated on Madagascar and made multiple long-distance invasions of oceanic islands including the Mascarenes, Aldabra, Comores, Seychelles, Andamans, and perhaps Pemba. The Mascarenes were probably colonised once, about 4.2-5.1Ma, and here Phelsuma rosagularis and Phelsuma inexpectata are newly recognised as species, as are three lineages of Phelsuma cepediana. Mascarene relationships are: Phelsuma edwardnewtoni, Phelsuma gigas (Phelsuma guentheri ((((P. cepediana A (P. cepediana B, C)) P. rosagularis) Phelsuma borbonica) ((Phelsuma ornata, P. inexpectata) Phelsuma guimbeaui))). The two recently extinct species, P. edwardnewtoni and the giant secondarily nocturnal Phelsuma gigas, differentiated on Rodrigues while on Mauritius the large nocturnal P. guentheri separated from a small diurnal form that radiated into six species, a likely result of volcanic activity. Two small-bodied lineages from Mauritius invaded the more recent island of Réunion producing two more species. Outside the Mascarenes, two mainly Madagascan assemblages are substantiated: Phelsuma serraticauda (Phelsuma lineata, Phelsuma laticauda, Phelsuma quadriocellata); (Phelsuma m. kochi (Phelsuma m. grandis, Phelsuma abboti)) (Phelsuma astriata, Phelsuma sundbergi). Their relationships to the Mascarene clade, and to Phelsuma mutabilis, Phelsuma standingi and Phelsuma andamanensis are unresolved.

Ancient mitochondrial DNA and morphology elucidate an extinct island radiation of Indian Ocean giant tortoises (Cylindraspis).

Ancient mitochondrial DNA sequences were used for investigating the evolution of an entire clade of extinct vertebrates, the endemic tortoises (Cylindraspis) of the Mascarene Islands in the Indian Ocean. Mitochondrial DNA corroborates morphological evidence that there were five species of tortoise with the following relationships: Cylindraspis triserrata ((Cylindraspis vosmaeri and Cylindraspis peltastes) (Cylindraspis inepta and Cylindraspis indica)). Phylogeny indicates that the ancestor of the group first colonized Mauritius where speciation produced C. triserrata and the ancestor of the other species including a second sympatric Mauritian form, C. inepta. A propagule derived from this lineage colonized Rodrigues 590 km to the east, where a second within-island speciation took place producing the sympatric C. vosmaeri and C. peltastes. A recent colonization of Réunion 150 km to the southwest produced C. indica. In the virtual absence of predators, the defensive features of the shells of Mascarene tortoises were largely dismantled, apparently in two stages. 'Saddlebacked' shells with high fronts evolved independently on all three islands. This and other features, such as a derived jaw structure and small body size, may be associated with niche differentiation in sympatric species and may represent a striking example of parallel differentiation in a large terrestrial vertebrate. The history of Mascarene tortoises contrasts with that of the Galápagos, where only a single species is present and surviving populations are genetically much more similar. However, they too show some reduction in anti-predator mechanisms and multiple development of populations with saddlebacked shells.

Problems of reproducibility--does geologically ancient DNA survive in amber-preserved insects?

Apparently ancient DNA has been reported from amber-preserved insects many millions of years old. Rigorous attempts to reproduce these DNA sequences from amber- and copal-preserved bees and flies have failed to detect any authentic ancient insect DNA. Lack of reproducibility suggests that DNA does not survive over millions of years even in amber, the most promising of fossil environments.

Palaeontology in a molecular world: the search for authentic ancient DNA.

The survival of ancient DNA in specimens up to several thousands of years old is established. However, there have been several claims concerning the recovery of geologically ancient DNA from fossil material many millions of years old. The authenticity of these fossil DNA sequences is questionable on theoretical and empirical grounds, and the existence of authentic geologically ancient DNA remains to be proven.

Novel Psychrobacter species from Antarctic ornithogenic soils.

Ornithogenic soil is derived from the deposition of the fecal matter of various species of birds and is a major source of nutrient input in the Antarctic marine ecosystem. A significant proportion of microbiota of ornithogenic soil collected from an Adélie penguin colony in eastern Antarctica (Vestfold Hills ice-free zone) consisted of gram-negative, coccoid bacteria identified on the basis of their phospholipid ester-linked fatty acid and lipid class profiles as Psychrobacter strains. Phenotypic, genotypic, and 16S ribosomal DNA phylogenetic analyses revealed that the Antarctic psychrobacters belonged to three distinct groups. Comparisons with Psychrobacter immobilis and Moraxella phenylpyruvica reference cultures isolated from fish, seawater, poultry, and human clinical specimens revealed the relationships of these groups within the genus Psychrobacter. Two of the groups represent the following two novel species: Psychrobacter urativorans sp. nov. (type strain, strain ACAM 534) and Psychrobacter frigidicola sp. nov. (type strain, strain ACAM 304). The third group of strains included members of the previously described species P. immobilis (Juni and Heym 1986). In addition, M. phenylpyruvica (Bøvre and Henriksen 1967) is renamed Psychrobacter phenylpyruvicus comb. nov. (type strain, strain ACAM 535) on the basis of 16S ribosomal DNA phylogenetic data. In general, the genus Psychrobacter could be differentiated from the related genera Moraxella and Acinetobacter by the fact that the members of the genus Psychrobacter are psychrotolerant or psychrophilic and halotolerant, which reflects the ubiquitous distribution of the genus in both marine and terrestrial environments. On the basis of the results of this and previous studies, the genus Psychrobacter is the predominant genus in ornithogenic soils in Antarctica and is diverse.

Molecular phylogenetics of Puffinus shearwaters: preliminary evidence from mitochondrial cytochrome b gene sequences.

Puffinus shearwaters (Family Procellariidae) are a diverse genus of pelagic seabirds or petrels with a worldwide distribution. Five subgroups of species have been recognized on the basis of osteological and external morphological characters. However, phylogenetic relationships among extant taxa are poorly understood, and evolutionary scenarios to explain the current overlapping distribution of the species subgroups are speculative. Phylogenetic analyses of partial mitochondrial cytochrome b gene sequences were used to examine the relationships among 19 species and subspecies of Puffinus shearwaters. In general, the molecular data support previous morphologically based phylogenetic hypotheses. However, the subgroup Neonectris appears to be polyphyletic with Puffinus nativitatis more closely related to species in the subgroup Puffinus. In addition the molecular data revealed a phylogenetic split between the Puffinus subgroup, with a worldwide distribution, and the remaining four subgroups which have an essentially southern hemisphere distribution. This suggests an evolutionary history in which the Puffinus ancestor was split between two geographic regions, from which dispersal and vicariant events resulted in the evolution and distribution of extant taxa.

Transaxillary endoscopic laser sympathectomy.

The authors describe a technique of achieving sympathetic denervation of the upper limbs in a 20-year-old woman with hyperhidrosis. A thoracoscope was inserted through a short incision in the axilla. A fibreoptic wave guide was passed through the thoracoscope to allow photocoagulation of the second thoracic sympathetic ganglion by Nd:YAG laser irradiation. The procedure was well tolerated and subsequent scar formation was unobtrusive.