An Integrated Biosecurity Risk Assessment Model (IBRAM) For Evaluating the Risk of Import Pathways for the Establishment of Invasive Species.

An important aspect of analyzing the risk of unwanted organisms establishing in an area is understanding the pathways by which they arrive. Evaluating the risks of these pathways requires use of data from multiple sources, which frequently are uncertain. To address the needs of agencies responsible for biosecurity operations, we present an Integrated Biosecurity Risk Assessment Model (IBRAM) for evaluating the risk of establishment and dispersal of invasive species along trade pathways. The IBRAM framework consists of multiple linked models which describe pest entry into the country, escape along trade pathways, initial dispersal into the environment, habitat suitability, probabilities of establishment and spread, and the consequences of these invasions. Bayesian networks (BN) are used extensively to model these processes. The model includes dynamic BN components and geographic data, resulting in distributions of output parameters over spatial and temporal axes. IBRAM is supported by a web-based tool that allows users to run the model on real-world pest examples and investigate the impact of alternative risk management scenarios, to explore the effect of various interventions and resource allocations. Two case studies are provided as examples of how IBRAM may be used: Queensland fruit fly (Bactrocera tryoni) (Diptera: Tephritidae) and brown marmorated stink bug (Halyomorpha halys) (Hemiptera: Pentatomidae) are unwanted organisms with the potential to invade Aotearoa New Zealand, and IBRAM has been influential in evaluating the efficacy of pathway management to mitigate the risk of their establishment in the country.

An 18-day, 3 °C cold treatment effectively kills Ceratitis capitata (Diptera: Tephritidae) in kiwifruit (Actinidia spp.).

A series of experiments were carried out to develop a phytosanitary disinfestation protocol to kill Ceratitis capitata (Weidemann) (Mediterranean fruit fly, Diptera: Tephritidae) in 'Hayward' kiwifruit (Actinidia deliciosa (A. Chev.) C.F. Liang and A.R. Ferguson) and 'Zesy002' kiwifruit (Actinidia chinensis Planch.). Experiments on 4 immature life stages (eggs and 3 larval instars) with treatment durations of between 5 and 18 days showed that third instars were the most tolerant to temperatures around 3 °C, with the lethal time to 99.9968% (probit 9) mortality (LT99.9968) estimated to be 17.3 days (95% confidence interval (CI) 16.4-18.2). Larvae reared and treated in 'Zesy002' were significantly more susceptible to cold treatment than those reared in 'Hayward'. A large-scale trial testing a disinfestation protocol of 3 ±â€…0.5 °C for 18 days treated over 500,000 third-instar C. capitata with no survivors. These results demonstrate that a cold treatment of 3.5 °C or below for 18 days induces C. capitata mortality in kiwifruit at a rate that exceeds 99.9968% with a degree of confidence greater than 99%.

Molecular phylogenetics of a South Pacific sap beetle species complex (Carpophilus spp., Coleoptera: Nitidulidae).

Several species of sap beetles in the genus Carpophilus are minor pests of fresh produce and stored products, and are frequently intercepted in biosecurity operations. In the South Pacific region, the superficially similar species C. maculatus and C. oculatus are frequently encountered in these situations. Three subspecies of C. oculatus have been described, and the complex of these four taxa has led to inaccurate identification and questions regarding the validity of these taxa. A molecular phylogenetic study using the mitochondrial gene cytochrome c oxidase I (COI) and two nuclear markers comprising the rDNA internal transcribed spacer 2 (ITS2) and the D1-D2 region of the large (28S) ribosomal RNA subunit showed that C. maculatus, and C. o. cheesmani were easily differentiated from the two other subspecies of C. oculatus. COI also showed differentiation between C. o. gilloglyi and C. o. oculatus, but this was not shown when third codon positions were removed and when RY-coding analyses were conducted. Generalised mixed Yule-Coalescent (GMYC) models were fitted to trees estimated from the COI data and were analysed using a multimodel approach to consider the evidence for three taxonomic groupings of the C. oculatus group. While the arrangement with the highest cumulative weight was not the arrangement ultimately accepted, the accepted taxonomy also had an acceptable level of support. ITS2 showed structure within C. oculatus, however C. o. oculatus was resolved as paraphyletic with respect to C. o. gilloglyi. COI showed evidence of sequence saturation and did not adequately resolve higher relationships between species represented in the dataset. 28S resolved higher relationships, but did not perform well at the species level. This study supports the validity of C. maculatus as a separate species, and provides sufficient evidence to raise C. o. cheesmani to the level of species. This study also shows significant structure within and between C. o. gilloglyi and C. o. oculatus, giving an indication of recent speciation events occurring. To highlight the interesting biology between these two taxa, C. o. gilloglyi is retained as a subspecies of C. oculatus. These results give clarity regarding the taxonomic status of C. maculatus and the subspecies of C. oculatus and provide a platform for future systematic research on Carpophilus.

A new species of Proterhinus Sharp, 1878 (Coleoptera: Curculionoidea: Belidae) from Miti'aro, Cook Islands, South Pacific.

Proterhinus tauai Brown, new species is described from specimens collected on the island of Miti'aro in the Cook Islands, South Pacific. It differs from congeners by the combination of having a dorsal vestiture of decumbent brown clavate scales, lacking erect elongate setae, anterior pronotal angles projecting forwards, and having short antennae that do not reach the posterior margin of the pronotum. This species shows strong sexual dimorphism in the shape of the rostrum, with males having a large semicircular rostrum, compared with a stout subrectangular rostrum in females. Fitting allometric models to the relationship of morphometric variables with body length show that rostrum width and area have significantly different allometric relationships between the sexes, consistent with hypotheses of sexual selection. Proterhinus tauai was collected from coastal scrub vegetation. Collection data and observation of captive specimens indicate a host relationship with the parasitic vine Cassytha filiformis L. (Lauraceae).

Redescription of Brachyolus punctatus White, 1846 (Coleoptera: Curculionidae: Entiminae), with synonyms and lectotype designations.

The New Zealand entimine weevil species Brachyolus punctatus White, 1846 is redescribed, with the following new synonyms proposed: Brachyolus albescens Broun, 1903, Inophloeus breviusculus Broun, 1880, Brachyolus elegans Broun, 1893 and Brachyolus viridescens Broun, 1893.

An annotated checklist of the Cook Islands psyllids with keys to the species and two new records (Hemiptera, Psylloidea).

An annotated checklist of the psyllids of the Cook Islands is presented. The presence of Syntomozatahuata (Klyver, 1932) and Triozaalifumosa Klyver, 1932 in the archipelago, based on new material collected, is reported for the first time. This is the first record from these islands of the genus Syntomoza and the family Liviidae. An identification key to the psyllid species known from the Cook Islands is provided, and their origin and provenance are discussed in relation to their biogeographic implications.

Austromonticola, a new genus of broad-nosed weevil (Coleoptera, Curculionidae, Entiminae) from montane areas of New Zealand.

Austromonticolagen. n. is proposed for a group of eight New Zealand alpine broad-nosed weevil species, all of which are here described: A. atriariussp. n. (type locality: Umbrella Mountains, Central Otago), A. caelibatussp. n. (type locality: Ohau Range, Mackenzie), A. furcatussp. n. (type locality: Old Man Range, Central Otago), A. inflatussp. n. (type locality: Hawkdun Range, Central Otago), A. planulatussp. n. (type locality: St Marys Range, Central Otago), A. postinventussp. n. (type locality: Kirkliston Range, South Canterbury), A. mataurasp. n. (type locality: Mt Dick, Otago Lakes) and A. rotundussp. n. (type locality: Old Man Range, Central Otago). All species occur exclusively above 1000 m elevation in the mountains of Central Otago and South Canterbury in the South Island. A phylogeny of the genus, including six outgroups, was inferred from 33 morphological characters. It resolved the genus as monophyletic, and revealed two strongly supported clades within Austromonticola. DNA sequences of four gene regions were obtained from five species. Of these, the 3' end of COI proved to be the most suitable for the identification of specimens. Females of all species have diagnostic secondary sexual structures on the elytra and ventrites. These structures are hypothesised to have evolved to assist with oviposition in and beside cushion plants or by selection for structures to mitigate the costs to females of prolonged mating.

A revision of the New Zealand weevil genus Irenimus Pascoe, 1876 (Coleoptera: Curculionidae: Entiminae).

The taxonomy of the New Zealand weevil genus Irenimus Pascoe, 1876 is revised, resulting in a narrower concept of the genus than has been considered in recent decades. In total, the genus now contains only seven species. In addition to the type species, I. parilis Pascoe, 1876, the genus contains I. duplex (Broun, 1904) and five newly described species: I. aniptus new species (type locality, Oamaru, DN), I. crinitus new species (type locality, Hakataramea Valley, SC), I. minimus new species (type locality, Alexandra, CO), I. stichus new species (type locality, Tekapo, MK) and I. thoracicus new species (type locality, Oamaru, DN). The genus Chalepistes new genus is established to contain the majority of species previously described in the genus Catoptes Schönherr, 1842, but also including species described in Brachyolus White, 1846; Irenimus Pascoe, 1876; Inophloeus Pascoe, 1875; and Nicaeana Pascoe, 1877. A total of 27 valid described species are new combinations with Chalepistes: C. aequalis (Broun, 1895) (from Irenimus), C. albosparsus (Broun, 1917) (from Irenimus), C. apicalis (Broun, 1923) (from Catoptes), C. asperatus (Broun, 1914) (from Brachyolus), C. compressus (Broun, 1880) (from Irenimus), C. costifer (Broun, 1886) (from Inophloeus), C. curvus (Barratt & Kuschel, 1996) (from Irenimus), C. dehiscens (Broun, 1917) (from Catoptes), C. dugdalei (Barratt & Kuschel, 1996) (from Irenimus), C. egens (Broun, 1904) (from Irenimus), C. inaequalis (Sharp, 1886) (from Brachyolus), C. instabilis (Marshall, 1931) (from Catoptes), C. latipennis (Broun, 1893) (from Catoptes), C. limbatus (Broun, 1909) (from Catoptes), C. lobatus (Broun, 1921) (from Catoptes), C. patricki (Barratt & Kuschel, 1996) (from Irenimus), C. pensus (Broun, 1914) (from Inophloeus), C. placidus (Broun, 1914) (from Nicaeana), C. posticalis (Broun, 1893) (from Irenimus), C. rhesus (Pascoe, 1875) (from Inophloeus), C. rubidus (Broun, 1881) (from Inophloeus), C. similis (Barratt & Kuschel, 1996) (from Irenimus), C. spectabilis (Broun, 1914) (from Catoptes), C. spermophilus (Broun, 1895), revised status (from Irenimus), C. stolidus (Broun, 1886) (from Irenimus), C. tenebricus (Broun, 1893) (from Catoptes), C. vastator (Broun, 1893) (from Irenimus). Numerous new synonyms with species of Chalepistes are also proposed: Brachyolus fuscipictus Broun, 1914 and Brachyolus terricola Broun, 1917 are junior subjective synonyms of Chalepistes asperatus (Broun); Brachyolus cervalis Broun, 1903 and Brachyolus sylvaticus Broun, 1910 are junior subjective synonyms of Chalepistes costifer (Broun); Inophloeus tricostatus Broun, 1915 is a junior subjective synonym of Chalepistes pensus (Broun); Catoptes pallidipes Broun, 1917, Catoptes flaviventris Broun, 1917 and Catoptes nigricans Broun, 1917 are junior subjective synonyms of Chalepistes placidus (Broun); Inophloeus longicornis Broun, 1904, Inophloeus medius Broun, 1893, Inophloeus sulcicollis Broun, 1914 and Inophloeus suturalis Broun, 1893 are junior subjective synonyms of Chalepistes rhesus (Pascoe); Inophloeus albonotata Broun, 1893, Catoptes asperellus Broun, 1893, Irenimus bicostatus Broun, 1886, Catoptes caliginosus Broun, 1893, Catoptes chalmeri Broun, 1893, Catoptes decorus Broun, 1893, Inophloeus discrepans Broun, 1904, Catoptes fumosus Broun, 1914, Catoptes furvus Broun, 1893, Catoptes humeralis Broun, 1893, Catoptes longulus Sharp, 1886, Inophloeus nigellus Broun, 1881, Irenimus pilosellus Broun, 1886 and Catoptes scutellaris Sharp, 1886 are junior subjective synonyms of Chalepistes rubidus (Broun); Catoptes subnitidus Broun, 1914 and Catoptes curvatus Broun, 1914 are junior subjective synonyms of Chalepistes spermophilus (Broun); Catoptes brevicornis Sharp, 1886 and Catoptes vexator Broun, 1904 are junior subjective synonyms of Chalepistes stolidus (Broun); and Catoptes aemulator Broun, 1893 and Catoptes argentalis Broun, 1914 are junior subjective synonyms of Chalepistes tenebricus (Broun). Additional new combinations include Inophloeus robustus (Broun, 1917) (from Catoptes) and Nicaeana fraudator (Marshall, 1931) (from Catoptes), while Catoptes postrectus Marshall, 1931 is a new synonym of Protolobus obscurus Sharp, 1886.

Barcoding and border biosecurity: identifying cyprinid fishes in the aquarium trade.

BACKGROUND: Poorly regulated international trade in ornamental fishes poses risks to both biodiversity and economic activity via invasive alien species and exotic pathogens. Border security officials need robust tools to confirm identifications, often requiring hard-to-obtain taxonomic literature and expertise. DNA barcoding offers a potentially attractive tool for quarantine inspection, but has yet to be scrutinised for aquarium fishes. Here, we present a barcoding approach for ornamental cyprinid fishes by: (1) expanding current barcode reference libraries; (2) assessing barcode congruence with morphological identifications under numerous scenarios (e.g. inclusion of GenBank data, presence of singleton species, choice of analytical method); and (3) providing supplementary information to identify difficult species. METHODOLOGY/PRINCIPAL FINDINGS: We sampled 172 ornamental cyprinid fish species from the international trade, and provide data for 91 species currently unrepresented in reference libraries (GenBank/Bold). DNA barcodes were found to be highly congruent with our morphological assignments, achieving success rates of 90-99%, depending on the method used (neighbour-joining monophyly, bootstrap, nearest neighbour, GMYC, percent threshold). Inclusion of data from GenBank (additional 157 spp.) resulted in a more comprehensive library, but at a cost to success rate due to the increased number of singleton species. In addition to DNA barcodes, our study also provides supporting data in the form of specimen images, morphological characters, taxonomic bibliography, preserved vouchers, and nuclear rhodopsin sequences. Using this nuclear rhodopsin data we also uncovered evidence of interspecific hybridisation, and highlighted unrecognised diversity within popular aquarium species, including the endangered Indian barb Puntius denisonii. CONCLUSIONS/SIGNIFICANCE: We demonstrate that DNA barcoding provides a highly effective biosecurity tool for rapidly identifying ornamental fishes. In cases where DNA barcodes are unable to offer an identification, we improve on previous studies by consolidating supplementary information from multiple data sources, and empower biosecurity agencies to confidently identify high-risk fishes in the aquarium trade.

Spider: an R package for the analysis of species identity and evolution, with particular reference to DNA barcoding.

Spider: SPecies IDentity and Evolution in R is a new R package implementing a number of useful analyses for DNA barcoding studies and associated research into species delimitation and speciation. Included are functions essential for generating important summary statistics from DNA barcode data, assessing specimen identification efficacy, and for testing and optimizing divergence threshold limits. In terms of investigating evolutionary and taxonomic questions, techniques for assessing diagnostic nucleotides and probability of reciprocal monophyly are also provided. Additionally, a sliding window function offers opportunities to analyse information across a gene, essential for marker design in degraded DNA studies. Spider capitalizes on R's extensible ethos and offers an integrated platform ideal for the analysis of both nucleotide and morphological data. The program can be obtained from the comprehensive R archive network (CRAN, http://cran.r-project.org) and from the R-Forge package development site (http://spider.r-forge.r-project.org/).

Sliding window analyses for optimal selection of mini-barcodes, and application to 454-pyrosequencing for specimen identification from degraded DNA.

DNA barcoding remains a challenge when applied to diet analyses, ancient DNA studies, environmental DNA samples and, more generally, in any cases where DNA samples have not been adequately preserved. Because the size of the commonly used barcoding marker (COI) is over 600 base pairs (bp), amplification fails when the DNA molecule is degraded into smaller fragments. However, relevant information for specimen identification may not be evenly distributed along the barcoding region, and a shorter target can be sufficient for identification purposes. This study proposes a new, widely applicable, method to compare the performance of all potential 'mini-barcodes' for a given molecular marker and to objectively select the shortest and most informative one. Our method is based on a sliding window analysis implemented in the new R package SPIDER (Species IDentity and Evolution in R). This method is applicable to any taxon and any molecular marker. Here, it was tested on earthworm DNA that had been degraded through digestion by carnivorous landsnails. A 100 bp region of 16 S rDNA was selected as the shortest informative fragment (mini-barcode) required for accurate specimen identification. Corresponding primers were designed and used to amplify degraded earthworm (prey) DNA from 46 landsnail (predator) faeces using 454-pyrosequencing. This led to the detection of 18 earthworm species in the diet of the snail. We encourage molecular ecologists to use this method to objectively select the most informative region of the gene they aim to amplify from degraded DNA. The method and tools provided here, can be particularly useful (1) when dealing with degraded DNA for which only small fragments can be amplified, (2) for cases where no consensus has yet been reached on the appropriate barcode gene, or (3) to allow direct analysis of short reads derived from massively parallel sequencing without the need for bioinformatic consolidation.