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Abstract Background The composition of the arthropod head is one of the most contentious issues in animal evolution. In particular, controversy surrounds the homology and innervation of segmental cephalic appendages by the brain. Onychophora (velvet worms) play a crucial role in understanding the evolution of the arthropod brain, because they are close relatives of arthropods and have apparently changed little since the Early Cambrian. However, the segmental origins of their brain neuropils and the number of cephalic appendages innervated by the brain - key issues in clarifying brain composition in the last common ancestor of Onychophora and Arthropoda - remain unclear. Results Using immunolabelling and neuronal tracing techniques in the developing and adult onychophoran brain, we found that the major brain neuropils arise from only the anterior-most body segment, and that two pairs of segmental appendages are innervated by the brain. The region of the central nervous system corresponding to the arthropod tritocerebrum is not differentiated as part of the onychophoran brain but instead belongs to the ventral nerve cords. Conclusions Our results contradict the assumptions of a tripartite (three-segmented) brain in Onychophora and instead confirm the hypothesis of bipartite (two-segmented) brain composition. They suggest that the last common ancestor of Onychophora and Arthropoda possessed a brain consisting of protocerebrum and deutocerebrum whereas the tritocerebrum evolved in arthropods.

Abstract Background In systems where two or more species experience secondary contact, behavioural factors that regulate interspecific gene flow may be important for maintaining species boundaries and reducing the incidence of hybridisation. At subantarctic Macquarie Island, two species of fur seal breed in close proximity to one another, hybridise at very high levels (up to 21% of hybrid pups are born annually), yet retain discrete gene pools. Using spatial and genetic information collected for pups and adults over twelve years, we assessed two behavioural traits - inter-annual site fidelity and differences in habitat use between the species - as possible contributors to the maintenance of this species segregation. Further, we explored the breakdown of these traits in pure-species individuals and hybrids. Results We found virtually complete spatial segregation of the parental species, with only one exception; a single territory that contained adults of both species and also the highest concentration of hybrid pups. The spatial distribution of each species was closely linked to habitat type (pebbled vs boulder beaches), with members of each species breeding almost exclusively on one type or the other but hybrids breeding on both or at the junction between habitats. Inter-annual site fidelity was high for both sexes of pure-species adults, with 66% of females and all males returning to the same territory or a neighbouring one in different years. An important consequence for pure females of breeding on the 'wrong' habitat type, and thus in a heterospecific aggregation, was the production of hybrid pups. Low habitat fidelity of hybrid females facilitated bi-directional backcrossing, resulting in more diverse hybrid offspring. Conclusion In a disturbed system where two sympatric fur seal species breed in close proximity, discrete gene pools are retained by extremely fine-scale and strong spatial segregation of the species. Two behavioural traits were found to be important in maintaining this stable population structure, and habitat type was a strong indicator of where species locate and a potentially powerful predictor of future directions of hybridisation. A direct consequence of the breakdown of this trait was the production of hybrid offspring, which may have severe implications if hybrids have reduced fitness.

Abstract Background A widely-used approach for screening nuclear DNA markers is to obtain sequence data and use bioinformatic algorithms to estimate which two alleles are present in heterozygous individuals. It is common practice to omit unresolved genotypes from downstream analyses, but the implications of this have not been investigated. We evaluated the haplotype reconstruction method implemented by PHASE in the context of phylogeographic applications. Empirical sequence datasets from five non-coding nuclear loci with gametic phase ascribed by molecular approaches were coupled with simulated datasets to investigate three key issues: (1) haplotype reconstruction error rates and the nature of inference errors, (2) dataset features and genotypic configurations that drive haplotype reconstruction uncertainty, and (3) impacts of omitting unresolved genotypes on levels of observed phylogenetic diversity and the accuracy of downstream phylogeographic analyses. Results We found that PHASE usually had very low false-positives (i.e., a low rate of confidently inferring haplotype pairs that were incorrect). The majority of genotypes that could not be resolved with high confidence included an allele occurring only once in a dataset, and genotypic configurations involving two low-frequency alleles were disproportionately represented in the pool of unresolved genotypes. The standard practice of omitting unresolved genotypes from downstream analyses can lead to considerable reductions in overall phylogenetic diversity that is skewed towards the loss of alleles with larger-than-average pairwise sequence divergences, and in turn, this causes systematic bias in estimates of important population genetic parameters. Conclusions A combination of experimental and computational approaches for resolving phase of segregating sites in phylogeographic applications is essential. We outline practical approaches to mitigating potential impacts of computational haplotype reconstruction on phylogeographic inferences. With targeted application of laboratory procedures that enable unambiguous phase determination via physical isolation of alleles from diploid PCR products, relatively little investment of time and effort is needed to overcome the observed biases.

Examines the microsatellite markers for the onychophoran Euperipatoides rowelli. Design of primers for dinucleotide regions; Optimization of primer pairs; Examination of loci by standard acrylamide gel isotopic microsatellite polymerase chain reaction.

Abstract Background Molecular genetic approaches have much to offer population biology. Despite recent advances, convenient techniques to develop and screen highly-resolving markers can be limiting for some applications and taxa. We describe an improved PCR-based, cloning-free, nuclear marker development procedure, in which single-stranded conformation polymorphism (SSCP) plays a central role. Sequence-variable alleles at putative nuclear loci are simultaneously identified and isolated from diploid tissues. Based on a multiple allele alignment, locus-specific primers are designed in conserved regions, minimizing 'null' alleles. Using two undescribed endemic Australian Collembola as exemplars, we outline a comprehensive approach to generating and validating suites of codominant, sequence-yielding nuclear loci for previously unstudied invertebrates. Results Six markers per species were developed without any baseline genetic information. After evaluating the characteristics of each new locus via SSCP pre-screening, population samples were genotyped on the basis of either DNA sequence, restriction site, or insertion/deletion variation, depending on which assay was deemed most appropriate. Polymorphism was generally high (mean of nine alleles per locus), and the markers were capable of resolving population structuring over very fine spatial scales ( Conclusion The comprehensive approach presented here feasibly overcomes technical hurdles of (i) developing suitably polymorphic nuclear loci for non-model organisms, (ii) physically isolating nuclear allele haplotypes from diploid tissues without cloning, and (iii) genotyping population samples on the basis of nuclear DNA sequence variation.