David C. Blackburn

DNA damage in preserved specimens and tissue samples: a molecular assessment

The extraction of genetic information from preserved tissue samples or museum specimens is a fundamental component of many fields of research, including the Barcode of Life initiative, forensic investigations, biological studies using scat sample analysis, and cancer research utilizing formaldehyde-fixed, paraffin-embedded tissue. Efforts to obtain genetic information from these sources are often hampered by an inability to amplify the desired DNA as a consequence of DNA damage.Previous studies have described techniques for improved DNA extraction from such samples or focused on the effect of damaging agents – such as light, oxygen or formaldehyde – on free nucleotides.We present ongoing work to characterize lesions in DNA samples extracted from preserved specimens. The extracted DNA is digested to single nucleosides with a combination of DNase I, Snake Venom Phosphodiesterase, and Antarctic Phosphatase and then analyzed by HPLC-ESI-TOF-MS.We present data for moth specimens that were preserved dried and pinned with no additional preservative and for frog tissue samples that were preserved in either ethanol, or formaldehyde, or fixed in formaldehyde and then preserved in ethanol. These preservation methods represent the most common methods of preserving animal specimens in museum collections. We observe changes in the nucleoside content of these samples over time, especially a loss of deoxyguanosine. We characterize the fragmentation state of the DNA and aim to identify abundant nucleoside lesions. Finally, simple models are introduced to describe the DNA fragmentation based on nicks and double-strand breaks.

Biogeography and evolution of body size and life history of African frogs: Phylogeny of squeakers (Arthroleptis) and long-fingered frogs (Cardioglossa) estimated from mitochondrial data

The evolutionary history of living African amphibians remains poorly understood. This study estimates the phylogeny within the frog genera Arthroleptis and Cardioglossa using approximately 2400 bases of mtDNA sequence data (12S, tRNA-Valine, and 16S genes) from half of the described species. Analyses are conducted using parsimony, maximum likelihood, and Bayesian methods. The effect of alignment on phylogeny estimation is explored by separately analyzing alignments generated with different gap costs and a consensus alignment. The consensus alignment results in species paraphyly, low nodal support, and incongruence with the results based on other alignments, which produced largely similar results. Most nodes in the phylogeny are highly supported, yet several topologies are inconsistent with previous hypotheses. The monophyly of Cardioglossa and of miniature species previously assigned to Schoutedenella was further examined using Templeton and Shimodaira-Hasegawa tests. Cardioglossa monophyly is rejected and C. aureoli is transferred to Arthroleptis. These tests do not reject Schoutedenella monophyly, but this hypothesis receives no support from non-parametric bootstrapping or Bayesian posterior probabilities. This phylogeny provides a framework for reconstructing historical biogeography and analyzing the evolution of body size and life history. Direct development and miniaturization appear at the base of Arthroleptis phylogeny concomitant with a range expansion from Central Africa to throughout most of sub-Saharan Africa.

Pax6 regulation of Math5 during mouse retinal neurogenesis.

Activation of the bHLH factor Math5 (Atoh7) is an initiating event for mammalian retinal neurogenesis, as it is critically required for retinal ganglion cell formation. However, the cis‐regulatory elements and trans‐acting factors that control Math5 expression are largely unknown. Using a combination of transgenic mice and bioinformatics, we identified a phylogenetically conserved regulatory element that is required to activate Math5 transcription during early retinal neurogenesis. This element drives retinal expression in vivo, in a cross‐species transgenic assay. Previously, Pax6 was shown to be necessary for the initiation of Math5 mRNA expression. We extend this finding by showing that the Math5 retinal enhancer also requires Pax6 for its activation, via Pax6 binding to a highly conserved binding site. In addition, our data reveal that other retinal factors are required for accurate regulation of Math5 by Pax6. genesis 47:175–187, 2009.

Evolution of arthropod visual systems: Development of the eyes and central visual pathways in the horseshoe crab Limulus polyphemus linnaeus, 1758 (Chelicerata, Xiphosura)

Despite ongoing interest into the architecture, biochemistry, and physiology of the visual systems of the xiphosuran Limulus polyphemus, their ontogenetic aspects have received little attention. Thus, we explored the development of the lateral eyes and associated neuropils in late embryos and larvae of these animals. The first external evidence of the lateral eyes was the appearance of white pigment spots—guanophores associated with the rudimentary photoreceptors—on the dorsolateral side of the late embryos, suggesting that these embryos can perceive light. The first brown pigment emerges in the eyes during the last (third) embryonic molt to the trilobite stage. However, ommatidia develop from this field of pigment toward the end of the larval trilobite stage so that the young larvae at hatching do not have object recognition. Double staining with the proliferation marker bromodeoxyuridine (BrdU) and an antibody against L. polyphemus myosin III, which is concentrated in photoreceptors of this species, confirmed previous reports that, in the trilobite larvae, new cellular material is added to the eye field from an anteriorly located proliferation zone. Pulse–chase experiments indicated that these new cells differentiate into new ommatidia. Examining larval eyes labeled for opsin showed that the new ommatidia become organized into irregular rows that give the eye field a triangular appearance. Within the eye field, the ommatidia are arranged in an imperfect hexagonal array. Myosin III immunoreactivity in trilobite larvae also revealed the architecture of the central visual pathways associated with the median eye complex and the lateral eyes. Double labeling with myosin III and BrdU showed that neurogenesis persists in the larval brain and suggested that new neurons of both the lamina and the medulla originate from a single common proliferation zone. These data are compared with eye development in Drosophila melanogaster and are discussed with regard to new ideas on eye evolution in the Euarthropoda. Developmental Dynamics 235:2641–2655, 2006.

Isolation and expression of Pax6 and atonal homologues in the American horseshoe crab, Limulus polyphemus

Pax6 regulates eye development in many animals. In addition, Pax6 activates atonal transcription factors in both invertebrate and vertebrate eyes. Here, we investigate the roles of Pax6 and atonal during embryonic development of Limulus polyphemus rudimentary lateral, medial and ventral eyes, and the initiation of lateral ommatidial eye and medial ocelli formation. Limulus eye development is of particular interest because these animals hold a unique position in arthropod phylogeny and possess multiple eye types. Furthermore, the molecular underpinnings of eye development have yet to be investigated in chelicerates. We characterized a Limulus Pax6 gene, with multiple splice products and predicted protein isoforms, and one atonal homologue. Unexpectedly, neither gene is expressed in the developing eye types examined, although both genes are present in the lateral sense organ, a structure of unknown function. Developmental Dynamics 237:2209–2219, 2008.

Ontogenetic Diet Change in the Arthroleptid Frog Schoutedenella xenodactyloides

Abstract Anuran amphibians are important consumers of arthropods in tropical ecosystems. Previous research has indicated that very small, terrestrial frogs, especially juveniles, largely consume small leaf litter arthropods. To date, few studies have examined diet in African anurans, and no studies exist of ontogenetic change in prey composition for any African frog. We investigated the change in diet that accompanies body size increase in the arthroleptid frog Schoutedenella xenodactyloides (Anura: Ranoidea) from a population located on the Mulanje Massif in Malawi, central Africa. Schoutedenella xenodactyloides is a miniature (< 22 mm snout–urostyle length; SUL), direct-developing frog that is often very abundant and is likely an important consumer of small leaf litter arthropods. Based on examination of stomach and intestinal contents from specimens that span the known range of posthatching body sizes, we document the taxonomic diversity of prey consumed by S. xenodactyloides. We present evidence that S. xenodactyloides exhibits a size-related ontogenetic change in the type and relative proportions of prey taxa. Small frogs (≤ 13 mm SUL) consume large numbers of collembolans and mites. As frogs attain larger body sizes; ants constitute a larger percentage of the total number of prey consumed; and collembolan and mite consumption falls below 10% of the total prey items. The prey consumed by S. xenodactyloides include at least 10 orders of arthropods and an unidentified mollusk. This is a greater diversity of prey items than previously found in Schoutedenella as well as most other arthroleptid species.

Cardioglossa liberiensis Barbour & Loveridge 1927 is a junior synonym of Phrynobatrachus fraterculus (Chabanaud 1921)

Abstract In 1927, Barbour & Loveridge described two species of Cardioglossa based on seven specimens from Liberia: C. decorata (six specimens) and C. liberiensis. Cardioglossa decorata has long been recognized as a junior synonym of Phrynobatrachus fraterculus (Chabanaud 1921), which is known from Guinea, Liberia, Sierra Leone, and Côte dIvoire. Cardioglossa liberiensis Barbour & Loveridge 1927, known from only a single specimen, is also a junior synonym of P. fraterculus (Chabanaud 1921). Cardioglossa liberiensis represents the seemingly rare maculiventris” colour phase of P. fraterculus that was first recognized by Guibé & Lamotte (1963). At present, only two species of Cardioglossa, C. aureoli and C. leucomystax, are known from west of Nigeria.

Concealed weapons: Erectile claws in African frogs

Vertebrate claws are used in a variety of important behaviours and are typically composed of a keratinous sheath overlying the terminal phalanx of a digit. Keratinous claws, however, are rare in living amphibians; their microstructure and other features indicate that they probably originated independently from those in amniotes. Here we show that certain African frogs have a different type of claw, used in defence, that is unique in design among living vertebrates and lacks a keratinous covering. These frogs have sectorial terminal phalanges on their hind feet that become functional by cutting through the skin. In the resting state, the phalanx is subdermal and attached to a distal bony nodule, a neomorphic skeletal element, via collagen-rich connective tissue. When erected, the claw breaks free from the nodule and pierces the ventral skin. The nodule, suspended by a sheath attached to the terminal phalanx and supported by collagenous connections to the dermis, remains fixed in place. While superficially resembling the shape of claws in other tetrapods, these are the only vertebrate claws known to pierce their way to functionality.

New Species of Arthroleptis (Anura: Arthroleptidae) from Ngozi Crater in the Poroto Mountains of Southwestern Tanzania

Abstract A new species of squeaker frog (Arthroleptis) is described from Ngozi Crater in the Poroto Mountains of southwestern Tanzania. The holotype, and only specimen, was collected 80 years ago but remained undescribed because of past taxonomic confusion relating to large-bodied species of Arthroleptis from eastern Africa. Morphological study, including multivariate analysis of measurement data, indicates that this new species is distinguishable from other species of Arthroleptis, including large-bodied species such as Arthroleptis adolfifriederici, Arthroleptis affinis, Arthroleptis nikeae, Arthroleptis nguruensis, and Arthroleptis tanneri. The new species differs from other described species of Arthroleptis by the combination of its large body size, relatively long toes, and relatively small tympanum. The description of this new species further highlights the biological diversity of the Southern Rift Mountains.

Diversity within diversity: Parasite species richness in poison frogs assessed by transcriptomics

Symbionts (e.g., endoparasites and commensals) play an integral role in their hosts ecology, yet in many cases their diversity is likely underestimated. Although endoparasites are traditionally characterized using morphology, sequences of conserved genes, and shotgun metagenomics, host transcriptomes constitute an underused resource to identify these organisms diversity. By isolating non-host transcripts from host transcriptomes, individual host tissues can now simultaneously reveal their endoparasite species richness (i.e., number of different taxa) and provide insights into parasite gene expression. These approaches can be used in host taxa whose endoparasites are mostly unknown, such as those of tropical amphibians. Here, we focus on the poison frogs (Dendrobatidae) as hosts, which are a Neotropical clade known for their bright coloration and defensive alkaloids. These toxins are an effective protection against vertebrate predators (e.g., snakes and birds), bacteria, and skin-biting ectoparasites (e.g., mosquitoes); however, little is known about their deterrence against eukaryotic endoparasites. With de novo transcriptomes of dendrobatids, we developed a bioinformatics pipeline for endoparasite identification that uses host annotated RNA-seq data and set of a priori parasite taxonomic terms, which are used to mine for specific endoparasites. We found a large community of helminths and protozoans that were mostly restricted to the digestive tract and a few systemic parasites (e.g., Trypanosoma). Contrary to our expectations, all dendrobatid frogs regardless of the presence of alkaloid defenses have endoparasites, with their highest species richness located in the frog digestive tract. Some of these organisms (e.g., roundworms) might prove to be generalists, as they were not found to be co-diversifying with their frog hosts. We propose that endoparasites may escape poison frogs chemical defenses by colonizing tissues with fewer alkaloids than the frogs skin, where most toxins are stored.