The seven deadly sins of DNA barcoding.

Despite the broad benefits that DNA barcoding can bring to a diverse range of biological disciplines, a number of shortcomings still exist in terms of the experimental design of studies incorporating this approach. One underlying reason for this lies in the confusion that often exists between species discovery and specimen identification, and this is reflected in the way that hypotheses are generated and tested. Although these aims can be associated, they are quite distinct and require different methodological approaches, but their conflation has led to the frequently inappropriate use of commonly used analytical methods such as neighbour-joining trees, bootstrap resampling and fixed distance thresholds. Furthermore, the misidentification of voucher specimens can also have serious implications for end users of reference libraries such as the Barcode of Life Data Systems, and in this regard we advocate increased diligence in the a priori identification of specimens to be used for this purpose. This commentary provides an assessment of seven deficiencies that we identify as common in the DNA barcoding literature, and outline some potential improvements for its adaptation and adoption towards more reliable and accurate outcomes.

Noninvasive recovery and detection of possum Trichosurus vulpecula DNA from bitten bait interference devices (WaxTags).

The brushtail possum is a major agricultural and ecological pest in New Zealand. A novel noninvasive DNA sampling tool for detecting its presence (WaxTags, or WT) was tested. DNA was recovered from saliva left on WT, and two lengths (407 bp and 648 bp) of the cytochrome c oxidase I (COI) barcoding region were amplified by polymerase chain reaction (PCR). PCR products were considered (+) when a DNA band was clearly visible by electrophoresis. Different factors that might affect PCR (+) were investigated with captive possums: (i) both extraction protocols of the QIAGEN DNeasy Blood and Tissue Kit, (ii) effect of an overnight or longer delay of up to 3 weeks before DNA extraction on both COI amplicons, and (iii) effect of the individual, order and magnitude of the bite. Extraction protocols were not significantly different. The effect of the overnight delay was not significant, and amplification of the short amplicon was significantly higher (100%) than for the long fragment (48%). After a two or 3-week delay, the short amplicon had 94% and 56% PCR (+), success rates, respectively. Individual, order and magnitude of a bite had no significant effect. The delay trial was repeated with WT from the wild, for which PCR (+) rate of the short amplicon was 63%, regardless of freshness. Four microsatellites were amplified from captive WT samples. We conclude that DNA from saliva traces can be recovered from WT, a potential new tool for noninvasive monitoring of possums and other wildlife.

Phylogenetic status and matrilineal structure of the biting midge, Culicoides imicola, in Portugal, Rhodes and Israel.

The biting midge Culicoides imicola Kieffer (Diptera: Ceratopogonidae) is the most important Old World vector of African horse sickness (AHS) and bluetongue (BT). Recent increases of BT incidence in the Mediterranean basin are attributed to its increased abundance and distribution. The phylogenetic status and genetic structure of C. imicola in this region are unknown, despite the importance of these aspects for BT epidemiology in the North American BT vector. In this study, analyses of partial mitochondrial cytochrome oxidase subunit I gene (COI) sequences were used to infer phylogenetic relationships among 50 C. imicola from Portugal, Rhodes, Israel, and South Africa and four other species of the Imicola Complex from southern Africa, and to estimate levels of matrilineal subdivision in C. imicola between Portugal and Israel. Eleven haplotypes were detected in C. imicola, and these formed one well-supported clade in maximum likelihood and Bayesian trees implying that the C. imicola samples comprise one phylogenetic species. Molecular variance was distributed mainly between Portugal and Israel, with no haplotypes shared between these countries, suggesting that female-mediated gene flow at this scale has been either limited or non-existent. Our results provide phylogenetic evidence that C. imicola in the study areas are potentially competent AHS and BT vectors. The geographical structure of the C. imicola COI haplotypes was concordant with that of BT virus serotypes in recent BT outbreaks in the Mediterranean basin, suggesting that population subdivision in its vector can impose spatial constraints on BT virus transmission.

Phylogenetic analysis of the mitochondrial cytochrome oxidase subunit I gene of five species of the Culicoides imicola species complex.

The phylogenetic status of members of the Culicoides imicola Kieffer (Diptera: Ceratopogonidae) species complex of haematophagous midges is unknown, and simple means to identify the members using all life stages are unavailable. In this study, the status of three confirmed (C. imicola s.s., C. bolitinos Meiswinkel and C. loxodontis Meiswinkel) and two provisional (C. tuttifrutti Meiswinkel and C. kwagga Meiswinkel) members of the complex from South Africa was assessed using phylogenetic analysis of partial DNA and amino acid sequences of the mitochondrial cytochrome oxidase subunit I (COI) gene. The four or five individuals of each species analysed contained one or two haplotypes each. Interspecific divergence was significant and characterized by strong A <--> T transversion bias. Phylogenetic trees constructed using neighbour-joining, parsimony and maximum likelihood showed each species to be distinct. Combinations of sites for two restriction enzymes in the COI sequences were species-specific and could form the basis of a diagnostic PCR assay.

Phylogenetic analysis of partial sequences of elongation factor 1alpha identifies major groups of lice (Insecta: Phthiraptera).

As a first attempt to use molecular data to resolve the relationships between the four suborders of lice and within the suborder Ischnocera, we sequenced a 347-bp fragment of the elongation factor 1alpha gene of 127 lice (Insecta: Phthiraptera) as well as outgroup taxa from the order Psocoptera. A number of well-supported monophyletic groups were found but the relationships among many of these groups could not be resolved. While it is probable that multiple substitutions at high divergences and ancient radiation over a short period of time have contributed to the problem, we attribute most of this lack of resolution to the high ratio of taxa to characters. Nevertheless, the sequence data unequivocally support a number of important relationships that are at variance with the conclusions of morphological taxonomy. These include the sister group relationship of Chelopistes and Oxylipeurus, two lice occupying different ecological niches on the same host, which have previously been assigned to different families. These results provide evidence in support of the hypothesis that lice have speciated in situ on the host in response to niche specialization and that this has given rise to convergent morphologies in the lice of different host groups which share similar ecological niches. We discuss our attempts to overcome the limitations of this large data set, including the use of leaf stability analysis, a new method for analyzing the stability of taxa in a phylogenetic tree, and examine a number of hypotheses of relationships based on both traditional taxonomy and host associations.

Pretreatment with controlled-release morphine for pain after hysterectomy.

In a double-blind randomised study, two dosing regimens for controlled-release morphine tablets were compared against placebo to ascertain the extent of prophylactic postoperative pain control in 51 women undergoing abdominal hysterectomy. One group of patients received controlled-release morphine every 12 h for 2 days before surgery, a second group received a single dose of controlled-release morphine 2 h before surgery and a third group received placebo. Patient-controlled analgesia system demands were compared for the first 38 h after surgery and 10-point pain scores and McGill pain questionnaires were compared for the first 6 postoperative days and at 6 weeks after surgery. During the first 2 days after surgery, patients reported high levels of pain which were similar in all groups. Pain scores on the third and fourth postoperative days were significantly lower in those who had a single pre-operative dose of controlled-release morphine compared with placebo and those who had been treated with-morphine every 12 h for 2 days (p = 0.043 and 0.024 for third and fourth day respectively). Patient-controlled analgesia demands were also fewer and less variable in those patients receiving the single dose of morphine 2 h before surgery. The study shows a beneficial analgesic effect of a single pre-operative dose of morphine, but shows no benefit for a more prolonged pre-operative dosing regimen.

Effect of i.v. omeprazole on the pH and volume of gastric contents before surgery.

Gastric pH and volume were measured in 87 patients who had received i.v. omeprazole (a new gastric antisecretory agent) 40 mg or placebo 1 or 3 h before surgery. Omeprazole increased the pH of gastric contents in the majority of patients. Administration 1 h before surgery produced greater gastric pH and smaller volume than administration 3 h before surgery. There was a reduction in the number of patients with a pH of less than 2.5 and a volume of 25 ml or more in the treated groups compared with the combined placebo groups. Omeprazole may have a role in prophylaxis against acid aspiration syndrome.

Fluid flow through dural puncture sites. An in vitro comparison of needle point types.

Leakage of artificial cerebrospinal fluid through human dura was measured in vitro after puncture by spinal needles. Fluid loss tailed off in all cases and ceased within 5 minutes in 10% of punctures made with a 22-gauge needle, 28% made with a 26-gauge and 65% made with a 29-gauge needle (p less than 0.05). The fluid loss was not reduced by alignment of the level of the needle parallel to the longitudinal direction of the fibres. Whitacre point needles of 22 gauge produced lower fluid loss than a 22-gauge Quincke point needle inserted across the fibres (p less than 0.05). Leakage rate was related to needle size, but not related to the alignment of a Quincke point. Little or no leakage occurred with 29-gauge needles.

The zymogram technique: isoenzyme patterns as an aid in Penicillium classification.

The zymogram technique, a means of visualizing isoenzymes and comparing differences between related species, is described. The technique has been applied to the very difficult Penicillium subgenus Penicillium with notable success. Results have reinforced some aspects of accepted taxonomies and provided a basis for re-examination of others. The zymogram technique should have broad applicability to other fungal genera.