Comparative phylogeographic studies of animals with low mobility and/or high habitat specificity remain rare, yet such organisms may hold fine-grained palaeoecological signal. Comparisons of multiple, codistributed species can elucidate major historical events. As part of a multitaxon programme, mitochondrial cytochrome oxidase I (COI) variation was analysed in two species of terrestrial flatworm, Artioposthia lucasi and Caenoplana coerulea. We applied coalescent demographic estimators and nested clade analysis to examine responses to past, landscape-scale, cooling-drying events in a model system of montane forest (Tallaganda). Correspondence of haplotype groups in both species to previously proposed microbiogeographic regions indicates at least four refuges from cool, dry conditions. The region predicted to hold the highest quality refuges (the Eastern Slopes Region), is indicated to have been a long-term refuge in both species, but so are several other regions. Coalescent analyses suggest that populations of A. lucasi are declining, while C. coerulea is expanding, although stronger population substructure in the former could yield similar patterns in the data. The differences in spatial and temporal genetic variation in the two species could be explained by differences in ecological attributes: A. lucasi is predicted to have lower dispersal ability but may be better able to withstand cold conditions. Thus, different contemporary population dynamics may reflect different responses to recent (Holocene) climate warming. The two species show highly congruent patterns of catchment-based local genetic endemism with one another and with previously studied slime-mould grazing Collembola.

Rhinoceros (rhino) numbers have dwindled substantially over the past century. As a result, three of the five species are now considered to be critically endangered, one species is vulnerable and one species is near-threatened. Poaching has increased dramatically over the past decade due to a growing demand for rhino horn products, primarily in Asia. Improved wildlife forensic techniques, such as validated tests for species identification of seized horns, are critical to aid current enforcement and prosecution efforts and provide a deterrent to future rhino horn trafficking. Here, we present an internationally standardized species identification test based on a 230 base pair cytochrome-b region. This test improves on previous nested PCR protocols and can be used for the discrimination of samples with <20pg of template DNA, thus suitable for DNA extracted from horn products. The assay was designed to amplify water buffalo samples, a common 'rhino horn' substitute, but to exclude human DNA, a common contaminant. Phylogenetic analyses using this partial cytochrome-b region resolved the five extant rhino species. Testing successfully returned a sequence and correct identification for all of the known rhino horn samples and vouchered rhino samples from museum and zoo collections, and provided species level identification for 47 out of 52 unknown samples from seizures. Validation and standardization was carried out across five different laboratories, in four different countries, demonstrating it to be an effective and reproducible test, robust to inter laboratory variation in equipment and consumables (such as PCR reagents). This is one of the first species identification tests to be internationally standardized to produce data for evidential proceedings and the first published validated test for rhinos, one of the flagship species groups of the illegal wildlife trade and for which forensic tools are urgently required. This study serves as a model for how species identification tests should be standardized and disseminated for wildlife forensic testing.

The development of Genotyping-By-Sequencing (GBS) technologies enables cost-effective analysis of large numbers of Single Nucleotide Polymorphisms (SNPs), especially in "non-model" species. Nevertheless, as such technologies enter a mature phase, biases and errors inherent to GBS are becoming evident. Here, we evaluated the performance of double digest Restriction enzyme Associated DNA (ddRAD) sequencing in SNP genotyping studies including high number of samples. Datasets of sequence data were generated from three marine teleost species (>5500 samples, >2.5 × 10 bases in total), using a standardized protocol. A common bioinformatics pipeline based on STACKS was established, with and without the use of a reference genome. We performed analyses throughout the production and analysis of ddRAD data in order to explore (i) the loss of information due to heterogeneous raw read number across samples; (ii) the discrepancy between expected and observed tag length and coverage; (iii) the performances of reference based vs. de novo approaches; (iv) the sources of potential genotyping errors of the library preparation/bioinformatics protocol, by comparing technical replicates. Our results showed use of a reference genome and a posteriori genotype correction improved genotyping precision. Individual read coverage was a key variable for reproducibility; variance in sequencing depth between loci in the same individual was also identified as an important factor and found to correlate to tag length. A comparison of downstream analysis carried out with ddRAD vs single SNP allele specific assay genotypes provided information about the levels of genotyping imprecision that can have a significant impact on allele frequency estimations and population assignment. The results and insights presented here will help to select and improve approaches to the analysis of large datasets based on RAD-like methodologies.

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