Verena E. Kutschera

Nuclear Genomic Sequences Reveal that Polar Bears Are an Old and Distinct Bear Lineage

Ancient Bears Polar bears are well known for adapting to their cold Arctic climate. Some recent studies, based on mitochondrial DNA, concluded that they are a relatively young species and that these adaptations occurred quite quickly. Although mitochondrial DNA is regularly used to estimate evolutionary history, it has some well-known drawbacks, including sex-biased dispersal and hybridization. Thus, Hailer et al. (p. 344) looked at neutral genetic data that are distributed more widely across the genome of a relatively large sample of polar, brown, and black bears. Consistent with fossil-based studies, the analysis reveals polar bears as a sister lineage to all brown bears, with an estimated divergence time of 300,000 to 900,000 years ago. Thus, polar bears are indeed of a more ancient lineage, and more recent estimates based on mitochondrial DNA are likely to have been affected by past hybridization with brown bear. Genomic analyses show that polar bears as a species are older and genetically more distinct than previously estimated. Recent studies have shown that the polar bear matriline (mitochondrial DNA) evolved from a brown bear lineage since the late Pleistocene, potentially indicating rapid speciation and adaption to arctic conditions. Here, we present a high-resolution data set from multiple independent loci across the nuclear genomes of a broad sample of polar, brown, and black bears. Bayesian coalescent analyses place polar bears outside the brown bear clade and date the divergence much earlier, in the middle Pleistocene, about 600 (338 to 934) thousand years ago. This provides more time for polar bear evolution and confirms previous suggestions that polar bears carry introgressed brown bear mitochondrial DNA due to past hybridization. Our results highlight that multilocus genomic analyses are crucial for an accurate understanding of evolutionary history.

Bears in a Forest of Gene Trees: Phylogenetic Inference Is Complicated by Incomplete Lineage Sorting and Gene Flow

Ursine bears are a mammalian subfamily that comprises six morphologically and ecologically distinct extant species. Previous phylogenetic analyses of concatenated nuclear genes could not resolve all relationships among bears, and appeared to conflict with the mitochondrial phylogeny. Evolutionary processes such as incomplete lineage sorting and introgression can cause gene tree discordance and complicate phylogenetic inferences, but are not accounted for in phylogenetic analyses of concatenated data. We generated a high-resolution data set of autosomal introns from several individuals per species and of Y-chromosomal markers. Incorporating intraspecific variability in coalescence-based phylogenetic and gene flow estimation approaches, we traced the genealogical history of individual alleles. Considerable heterogeneity among nuclear loci and discordance between nuclear and mitochondrial phylogenies were found. A species tree with divergence time estimates indicated that ursine bears diversified within less than 2 My. Consistent with a complex branching order within a clade of Asian bear species, we identified unidirectional gene flow from Asian black into sloth bears. Moreover, gene flow detected from brown into American black bears can explain the conflicting placement of the American black bear in mitochondrial and nuclear phylogenies. These results highlight that both incomplete lineage sorting and introgression are prominent evolutionary forces even on time scales up to several million years. Complex evolutionary patterns are not adequately captured by strictly bifurcating models, and can only be fully understood when analyzing multiple independently inherited loci in a coalescence framework. Phylogenetic incongruence among gene trees hence needs to be recognized as a biologically meaningful signal.

Autofluorescence imaging, an excellent tool for comparative morphology

Here we present a set of methods for documenting (exo‐)morphology by applying autofluorescence imaging. For arthropods, but also for other taxa, autofluorescence imaging combined with composite imaging is a fast documentation method with high‐resolution capacities. Compared to conventional micro‐ and macrophotography, the illumination is much more homogenous, and structures are often better contrasted. Applying different wavelengths to the same object can additionally be used to enhance distinct structures. Autofluorescence imaging can be applied to dried and embedded specimens, but also directly on specimens within their storage liquid. This has an enormous potential for the documentation of rare specimens and especially type specimens without the need of preparation. Also for various fossils, autofluorescence can be used to enhance the contrast between the fossil and the matrix significantly, making even smallest details visible. ‘Life‐colour’ fluorescence especially is identified as a technique with great potential. It provides additional information for which otherwise more complex methods would have to be applied. The complete range of differences and variations between fluorescence macrophotography and different types of fluorescence microscopy techniques are here explored and evaluated in detail. Also future improvements are suggested. In summary, autofluorescence imaging is a powerful, easy and fast‐to‐apply tool for morphological studies.

Imaging and Documenting Gammarideans

We give an overview of available techniques for imaging and documenting applied to gammarideans and discuss their advantages and disadvantages. Although recent techniques, such as confocal laser scanning microscopy (cLSM), focused ion beam scanning electron microscopy (FIB SEM), or computed microtomography (μCT), provide new possibilities to detect and document structures, these high-tech devices are expensive, and access to them is often limited. Alternatively, there are many possibilities to enhance the capabilities of established techniques such as macrophotography and light microscopy. We discuss improvements of the illumination with polarized light and the possibilities of utilizing the autofluorescence of animals such as the gammarideans. In addition, we present software-based enhancing tools such as image fusion and image stitching.

Evolution of mantis shrimps (Stomatopoda, Malacostraca) in the light of new Mesozoic fossils

BackgroundWe describe new specimens of Mesozoic mantis shrimps (Stomatopoda, Malacostraca) that exhibit morphological and developmental information previously unknown.ResultsSpecimens assigned to the taxon Sculda exhibit preserved pleopods, thoracopods including all four raptorial limbs as well as details of antennae and antennulae. The pleopods and the antennulae resemble those of the modern mantis shrimps, but the raptorial limbs are not as differentiated as in the modern species. In some specimens, the first raptorial limb (second thoracopod) is not significantly larger than the similar-sized posterior three pairs (as in extant species), but instead these appendages become progressively smaller along the series. In this respect they resemble certain Palaeozoic stomatopods. Another specimen, most likely belonging to another species, has one pair of large anterior raptorial thoracopods, a median-sized pair and two more pairs of small-sized raptorial appendages and, thus, shows a new, previously unknown type of morphology. A single specimen of Pseudosculda laevis also exhibits the size of the raptorial limbs; they are differentiated as in modern species, one large pair and three small pairs. Furthermore, we report additional larval specimens and show also post-larval changes, e.g., of the tail fan.ConclusionsThese new data are used to reconsider the phylogeny of Stomatopoda. We still need a strict taxonomical revision of the Mesozoic mantis shrimps, but this first examination already demonstrates the importance of these fossils for understanding mantis shrimp evolution and the interpretation of evolutionary pathways of particular features.

A range-wide synthesis and timeline for phylogeographic events in the red fox (Vulpes vulpes)

BackgroundMany boreo-temperate mammals have a Pleistocene fossil record throughout Eurasia and North America, but only few have a contemporary distribution that spans this large area. Examples of Holarctic-distributed carnivores are the brown bear, grey wolf, and red fox, all three ecological generalists with large dispersal capacity and a high adaptive flexibility. While the two former have been examined extensively across their ranges, no phylogeographic study of the red fox has been conducted across its entire Holarctic range. Moreover, no study included samples from central Asia, leaving a large sampling gap in the middle of the Eurasian landmass.ResultsHere we provide the first mitochondrial DNA sequence data of red foxes from central Asia (Siberia), and new sequences from several European populations. In a range-wide synthesis of 729 red fox mitochondrial control region sequences, including 677 previously published and 52 newly obtained sequences, this manuscript describes the pattern and timing of major phylogeographic events in red foxes, using a Bayesian coalescence approach with multiple fossil tip and root calibration points. In a 335xa0bp alignment we found in total 175 unique haplotypes. All newly sequenced individuals belonged to the previously described Holarctic lineage. Our analyses confirmed the presence of three Nearctic- and two Japan-restricted lineages that were formed since the Mid/Late Pleistocene.ConclusionsThe phylogeographic history of red foxes is highly similar to that previously described for grey wolves and brown bears, indicating that climatic fluctuations and habitat changes since the Pleistocene had similar effects on these highly mobile generalist species. All three species originally diversified in Eurasia and later colonized North America and Japan. North American lineages persisted through the last glacial maximum south of the ice sheets, meeting more recent colonizers from Beringia during postglacial expansion into the northern Nearctic. Both brown bears and red foxes colonized Japan’s northern island Hokkaido at least three times, all lineages being most closely related to different mainland lineages. Red foxes, grey wolves, and brown bears thus represent an interesting case where species that occupy similar ecological niches also exhibit similar phylogeographic histories.

A sensitive and specific multiplex PCR approach for sex identification of ursine and tremarctine bears suitable for non-invasive samples

We report a new approach for molecular sex identification of extant Ursinae and Tremarctinae bears. Two Y‐specific fragments (SMCY and 318.2) and one X‐specific fragment (ZFX) are amplified in a multiplex PCR, yielding a double test for male‐specific amplification and an internal positive control. The primers were designed and tested to be bear‐specific, thereby minimizing the risk of cross‐amplification in other species including humans. The high sensitivity and small amplicon sizes (100, 124, 160 base pairs) facilitate analysis of non‐invasively obtained DNA material. DNA from tissue and blood as well as from 30 non‐invasively collected hair and faeces yielded clear and easily interpretable results. The fragments were detected both by standard gel electrophoresis and automated capillary electrophoresis.

Response to Comment on “Nuclear Genomic Sequences Reveal that Polar Bears Are an Old and Distinct Bear Lineage”

Nakagome et al. reanalyzed some of our data and assert that we cannot refute the mitochondrial DNA–based scenario for polar bear evolution. Their single-locus test statistic is strongly affected by introgression and incomplete lineage sorting, whereas our multilocus approaches are better suited to recover the true species relationships. Indeed, our sister-lineage model receives high support in a Bayesian model comparison.

Genetic signatures of adaptation revealed from transcriptome sequencing of Arctic and red foxes.

BackgroundThe genus Vulpes (true foxes) comprises numerous species that inhabit a wide range of habitats and climatic conditions, including one species, the Arctic fox (Vulpes lagopus) which is adapted to the arctic region. A close relative to the Arctic fox, the red fox (Vulpes vulpes), occurs in subarctic to subtropical habitats. To study the genetic basis of their adaptations to different environments, transcriptome sequences from two Arctic foxes and one red fox individual were generated and analyzed for signatures of positive selection. In addition, the data allowed for a phylogenetic analysis and divergence time estimate between the two fox species.ResultsThe de novo assembly of reads resulted in more than 160,000 contigs/transcripts per individual. Approximately 17,000 homologous genes were identified using human and the non-redundant databases. Positive selection analyses revealed several genes involved in various metabolic and molecular processes such as energy metabolism, cardiac gene regulation, apoptosis and blood coagulation to be under positive selection in foxes. Branch site tests identified four genes to be under positive selection in the Arctic fox transcriptome, two of which are fat metabolism genes. In the red fox transcriptome eight genes are under positive selection, including molecular process genes, notably genes involved in ATP metabolism. Analysis of the three transcriptomes and five Sanger re-sequenced genes in additional individuals identified a lower genetic variability within Arctic foxes compared to red foxes, which is consistent with distribution range differences and demographic responses to past climatic fluctuations. A phylogenomic analysis estimated that the Arctic and red fox lineages diverged about three million years ago.ConclusionsTranscriptome data are an economic way to generate genomic resources for evolutionary studies. Despite not representing an entire genome, this transcriptome analysis identified numerous genes that are relevant to arctic adaptation in foxes. Similar to polar bears, fat metabolism seems to play a central role in adaptation of Arctic foxes to the cold climate, as has been identified in the polar bear, another arctic specialist.

Re-evaluation of the Mesozoic mantis shrimp Ursquilla yehoachi based on new material and the virtual peel technique

We re-evaluate the Mesozoic mantis shrimp Ursquilla yehoachi, based on the original four specimens and a new exquisitely preserved one. All specimens were documented by application of a new technique, which is introduced here and termed the virtual peel technique. This technique includes two steps: 1) Documentation of lowrelief fossils including their 3D information with the aid of a standard flat-bed scanner. 2) Virtual inversion of the negative relief of fossils; the resulting positive relief facilitates a better understanding of the fossil. Unlike the previously known specimens, the new specimen of U. yehoachi preserved most details of the uropod including the exopod, an important feature in stomatopod phylogeny. Remarkably, this exopod bears striking similarities to that of modern stomatopods in being bipartite and having a paddleshaped distal part, supporting earlier assumptions about the systematic position of U. yehoachi within Squilloidea. Observable morphological differences among the available material of U. yehoachi can now be identified as ontogenetic variation. The telson, for example, attains a proportionally broader shape with increasing size, a result of allometric growth. Carolin Haug. Department of Cytology and Evolutionary Biology, Zoological Institute and Museum, University of Greifswald, Soldmannstrasse 23, 17487 Greifswald, Germany carolin.haug@palaeo-evodevo.info Verena Kutschera. Biosystematic Documentation, University of Ulm, Helmholtzstrasse 20, 89081 Ulm, Germany verena.kutschera@uni-ulm.de Shane T. Ahyong. Australian Museum, 6 College Street, Sydney, NSW 2010, Australia Shane.Ahyong@austmus.gov.au Francisco J. Vega. Instituto de Geología, UNAM, Ciudad Universitaria, Coyoacán, México DF 04510, Mexico vegver@unam.mx Andreas Maas. Biosystematic Documentation, University of Ulm, Helmholtzstrasse 20, 89081 Ulm, Germany andreas.maas@uni-ulm.de Dieter Waloszek. Biosystematic Documentation, University of Ulm, Helmholtzstrasse 20, 89081 Ulm, Germany dieter.waloszek@uni-ulm.de HAUG ET AL.: URSQUILLA AND VIRTUAL PEEL 2 Joachim T. Haug. Department of Cytology and Evolutionary Biology, Zoological Institute and Museum, University of Greifswald, Soldmannstrasse 23, 17487 Greifswald, Germany joachim.haug@palaeo-evodevo.info