Detecting and Removing Sample Contamination in Phylogenomic Data: An Example and its Implications for Cicadidae Phylogeny (Insecta: Hemiptera).

Contamination of a genetic sample with DNA from one or more nontarget species is a continuing concern of molecular phylogenetic studies, both Sanger sequencing studies and next-generation sequencing studies. We developed an automated pipeline for identifying and excluding likely cross-contaminated loci based on the detection of bimodal distributions of patristic distances across gene trees. When contamination occurs between samples within a data set, a comparison between a contaminated sample and its contaminant taxon will yield bimodal distributions with one peak close to zero patristic distance. This new method does not rely on a priori knowledge of taxon relatedness nor does it determine the causes(s) of the contamination. Exclusion of putatively contaminated loci from a data set generated for the insect family Cicadidae showed that these sequences were affecting some topological patterns and branch supports, although the effects were sometimes subtle, with some contamination-influenced relationships exhibiting strong bootstrap support. Long tip branches and outlier values for one anchored phylogenomic pipeline statistic (AvgNHomologs) were correlated with the presence of contamination. While the anchored hybrid enrichment markers used here, which target hemipteroid taxa, proved effective in resolving deep and shallow level Cicadidae relationships in aggregate, individual markers contained inadequate phylogenetic signal, in part probably due to short length. The cleaned data set, consisting of 429 loci, from 90 genera representing 44 of 56 current Cicadidae tribes, supported three of the four sampled Cicadidae subfamilies in concatenated-matrix maximum likelihood (ML) and multispecies coalescent-based species tree analyses, with the fourth subfamily weakly supported in the ML trees. No well-supported patterns from previous family-level Sanger sequencing studies of Cicadidae phylogeny were contradicted. One taxon (Aragualna plenalinea) did not fall with its current subfamily in the genetic tree, and this genus and its tribe Aragualnini is reclassified to Tibicininae following morphological re-examination. Only subtle differences were observed in trees after the removal of loci for which divergent base frequencies were detected. Greater success may be achieved by increased taxon sampling and developing a probe set targeting a more recent common ancestor and longer loci. Searches for contamination are an essential step in phylogenomic analyses of all kinds and our pipeline is an effective solution. [Auchenorrhyncha; base-composition bias; Cicadidae; Cicadoidea; Hemiptera; phylogenetic conflict.].

Culicoides species as potential vectors of African horse sickness virus in the southern regions of South Africa.

African horse sickness (AHS), a disease of equids caused by the AHS virus, is of major concern in South Africa. With mortality reaching up to 95% in susceptible horses and the apparent reoccurrence of cases in regions deemed non-endemic, most particularly the Eastern Cape, epidemiological research into factors contributing to the increase in the range of this economically important virus became imperative. The vectors, Culicoides (Diptera: Ceratopogonidae), are considered unable to proliferate during the unfavourable climatic conditions experienced in winter in the province, although the annual occurrence of AHS suggests that the virus has become established and that vector activity continues throughout the year. Surveillance of Culicoides within the province is sparse and little was known of the diversity of vector species or the abundance of known vectors, Culicoides imicola and Culicoides bolitinos. Surveillance was performed using light trapping methods at selected sites with varying equid species over two winter and two outbreak seasons, aiming to determine diversity, abundance and vector epidemiology of Culicoides within the province. The research provided an updated checklist of Culicoides species within the Eastern Cape, contributing to an increase in the knowledge of AHS vector epidemiology, as well as prevention and control in southern Africa.

Bloodmeal analysis in Culicoides midges collected near horses, donkeys and zebras in the Eastern Cape, South Africa.

An upsurge in African horse sickness (AHS) in the Eastern Cape, South Africa, from 2006 led to an epidemiological reassessment of the disease there. Light trapping surveys carried out near horses, donkeys and zebras in 2014-2016 collected 39 species of Culicoides midge (Diptera: Ceratopogonidae) that are potential vectors of AHS. To establish if these midges fed on equids, DNA sequences were obtained from the gut contents of 52 female midges (35 freshly blood-fed, 13 gravid and four parous), representing 11 species collected across 11 sites. Culicoides leucostictus fed on all three equids. Culicoides bolitinos, Culicoides imicola and Culicoides magnus fed on both horses and donkeys. Culicoides onderstepoortensis fed on donkeys, and Culicoides similis and Culicoides pycnostictus fed on zebras. Bloodmeals from cows, pigs, warthogs, impalas and a domestic dog were also identified in various species, but none of the midges tested had fed on birds. These results contribute to knowledge of the vectorial capacity of several species of Culicoides with regard to AHS in the Eastern Cape and point to potential reservoir hosts, of which donkeys, zebras and domestic dogs have previously been found to harbour AHS. Blood-fed midges were also obtained throughout winter, indicating the potential for endemic AHS in the province.

Advantages of using development models of the carrion beetles Thanatophilus micans (Fabricius) and T. mutilatus (Castelneau) (Coleoptera: Silphidae) for estimating minimum post mortem intervals, verified with case data.

Some beetles are as useful as blow flies for estimating the minimum post mortem interval (PMImin) or time since death. Examples include Thanatophilus micans (Fabricius) and Thanatophilus mutilatus (Castelneau), two geographically and ecologically overlapping African beetles. Molecular means of identifying these species, descriptions of their natural history, thermal summation models for the development of each species, and a case in which T. micans was recovered are presented. These beetles colonise bodies soon after death, their development spans more time than that of flies, and they may be little affected by maggot-generated heat. From an experimental perspective, they can be reared individually, which allows the identification of sick individuals and has analytical advantages relative to fly larvae that must be reared in groups. Estimating minimum post mortem intervals for both species using the case data strongly suggests that developmental models parameterised for one species should not be used to make forensic estimates for closely related species for which no specific model is available and emphasises the need for correct identifications.

Molecular phylogenetics of Oestroidea (Diptera: Calyptratae) with emphasis on Calliphoridae: insights into the inter-familial relationships and additional evidence for paraphyly among blowflies.

The superfamily Oestroidea, comprising ∼15,000 species, is a large and ecologically diverse clade within the order Diptera. Among its six commonly recognized families, Calliphoridae seems to be crucial for understanding evolutionary relationships in the group, as it is recognized as a controversial paraphyletic grouping. To further investigate this matter, the ITS2, 28S, COI and 16S regions were used to infer phylogenetic relationships in Oestroidea with maximum-parsimony (MP), maximum-likelihood (ML) and Bayesian inference (BI) methods. For the BI analyses, a deep evaluation of different data partitioning strategies was conducted, including consideration of structural conformation (ITS2 and 16S) and codon position (COI) information. Results suggest the existence of two main clades in Oestroidea: (Tachinidae+Mesembrinellinae) and (Rhiniinae, (Sarcophagidae+Calliphoridae sensu stricto)). Oestridae was recovered as sister group of the remaining Oestroidea in the MP trees while it was placed closer to the (Rhiniinae+Sarcophagidae+Calliphoridae sensu stricto) group in the ML and BI trees. A paraphyletic Calliphoridae was recovered, confirming the exclusion of Rhiniinae, a clade recently promoted to family status and therefore already excluded. Mesembrinellinae could also be considered a distinct group apart from Calliphoridae, although further studies are required. Consideration of structural and codon position information led to a significant increase in the log-likelihoods of the analyses, which were accompanied by small changes in the inferred topologies, branch lengths and posterior probability support values. However, as model complexity increases, so does uncertainty across the estimated parameters, including tree topologies, and phylogenies inferred under very parameter-rich models may be less reliable even when possessing higher log-likelihoods.

Amplified fragment length polymorphism confirms reciprocal monophyly in Chrysomya putoria and Chrysomya chloropyga: a correction of reported shared mtDNA haplotypes.

Reinvestigation of mitochondrial haplotypes previously reported to be shared between the Afrotropical blowflies Chrysomya putoria Weidemann and Chrysomya chloropyga Weidemann (Diptera: Calliphoridae) revealed an error resulting from the misidentification of specimens. Preliminary amplified fragment length polymorphism (AFLP) analysis of the original and additional individuals again failed to find reciprocal monophyly, leading to a re-examination of the specimens for diagnostic male genitalic characters that were first described following the earlier study. Four of the original study specimens were found to have been misidentified, and definitive analysis of both mtDNA and AFLP genotypes using phylogenetic analysis and genetic assignment showed that each species was indeed reciprocally monophyletic. In addition to correcting the earlier error, this study illustrates how AFLP analysis can be used for efficient and effective specimen identification through both phylogenetic analysis and genetic assignment, and suggests that the latter method has special advantages for identification when no conspecific specimens are represented in the reference database.

A watershed study on genetic diversity: phylogenetic analysis of the Platypleura plumosa (Hemiptera: Cicadidae) complex reveals catchment-specific lineages.

Historical biogeography studies have at their disposal a small suite of vicariance models to explain genetic differentiation within and between species. One of these processes involves the role of river catchments and their associated watersheds, in driving diversification and is applicable to both aquatic and terrestrial organisms. Although the idea of catchments structuring the genetic history of aquatic organisms is reasonably well understood, their effect on terrestrial organisms has largely been overlooked, with relevant studies being limited in scope. South Africa presents a perfect test-bed for elucidating this mechanism of diversification due to its rich biodiversity, range of climatic environments and many large river catchments. Here we use the cicadas of the Platypleura plumosa complex to highlight the importance of catchments and their associated watersheds in driving diversification of terrestrial invertebrates that lack an aquatic life-stage. Population structure was found to correspond to primary and in some cases secondary catchments; highlighting the need to include information on catchment structure when formulating hypotheses of population diversification. Recognizing that climate change in the near future is likely to alter the environment, and particularly precipitation patterns, insight into recent patterns of population change related to catchments may be useful in a conservation context.

Data quality in thermal summation development models for forensically important blowflies.

To highlight some issues regarding data quality that are significant in estimating post-mortem intervals (PMI) from maggots, the developmental constants of thermal summation models for development of Chrysomya megacephala Fabricius (Diptera: Calliphoridae) were calculated from incidental data gathered from 12 published studies, and from data generated specifically for the purpose in a single experiment. The focused experiment involved measuring the timing of five developmental landmarks at nine constant temperatures with a sampling resolution of 6-12 h, which is characteristic of other published studies. Combining data from different studies produced inconsistent results because of statistical noise introduced by (at least) disparities in temporal precision, descriptive statistics, geographical location and rearing diets. A robust experimental design to estimate a developmental model should involve at least six constant temperatures, starting at about 7 degrees C above the relevant developmental zero (D0) and going almost to the upper critical temperature, and a temporal sampling interval with a relative precision of about 10%, which requires sampling about every 2 h until hatching, about every 3 h until first ecdysis and about every 6 h until second ecdysis.

Models of development for blowfly sister species Chrysomya chloropyga and Chrysomya putoria.

Developmental curves for the sister species Chrysomya chloropyga (Wiedemann, 1818) and Chrysomya putoria (Wiedemann, 1830) (Diptera: Calliphoridae) were established at eight and 10 different constant temperatures, respectively, using developmental landmarks and body length as measures of age. The thermal summation constants (K) and developmental threshold (D(0)) were calculated for five developmental landmarks using a previously described method. Isomorphen and isomegalen diagrams were also constructed for the purpose of estimating postmortem intervals (PMIs). Chrysomya chloropyga had an average developmental threshold value (D(0)) of 10.91 degrees C (standard error [SE] = 0.94 degrees C, n = 5), significantly lower than that of C. putoria (13.42 degrees C, SE = 0.45 degrees C, n = 5) (paired t-test: t = - 4.63, d.f. = 8, P < 0.00). Similarly, K values for C. chloropyga were larger than those for C. putoria for all developmental events except onset of the wandering phase. These are the first data that can be used to calculate minimum PMIs and predict population growth of C. chloropyga and C. putoria in Africa; the data indicate that developmental data for one of these species cannot be used as surrogate data for the sister species.

Flies in the ointment: a morphological and molecular comparison of Lucilia cuprina and Lucilia sericata (Diptera: Calliphoridae) in South Africa.

Complementary nuclear (28S rRNA) and mitochondrial (COI) genes were sequenced from blowflies that phenotypically resembled Lucilia cuprina (W.), Lucilia sericata (Meigen) or exhibited characters of both species. The aim was to test a long-held hypothesis that these species hybridize under natural conditions in South Africa (Ullyett, 1945). Blowflies were obtained predominantly from the Cape Town metropolitan area, but reference samples were acquired for L. sericata from Pretoria. Several L. cuprina-like flies were shown to possess a conflicting combination of nuclear and mitochondrial genes that has also been seen in Hawaiian specimens. Homoplasy, sampling of pseudogenes, hybridization and incomplete lineage sorting are discussed as possible hypotheses for the pattern and the latter is concluded to represent the most likely explanation.

Spatial and Seasonal Distribution of Forensically Important Blow Flies (Diptera: Calliphoridae) in Makhanda, Eastern Cape, South Africa.

The seasonal activity of adults of eight forensically important blow fly species [Calliphora croceipalpis (Diptera: Calliphoridae), Jaennicke, Lucilia sericata (Meigen) (Diptera: Calliphoridae), L. cuprina (Wiedemann) (Diptera: Calliphoridae), Chrysomya chloropyga (Wiedemann) (Diptera: Calliphoridae), Ch. albiceps (Wiedemann) (Diptera: Calliphoridae), Ch. marginalis (Wiedemann) (Diptera: Calliphoridae), Ch. putoria (Wiedemann) (Diptera: Calliphoridae), Ch. megacephala (Fabricius) (Diptera: Calliphoridae)] was quantified from collections in Makhanda, South Africa. Flies were collected in traps baited with chicken liver and placed in the field at eight locations for four consecutive days each fortnight for 14 mo. The seasonal distribution of each species compared well to published seasonal distributions of these blow flies elsewhere in South Africa, with evidence of year-to-year variation within seasons that might be explained by weather. This information is important for determining when and where certain species are likely to occur and will be of use in forensic investigations and myiasis management plans.

Baited traps may be an alternative to conventional pesticides in integrated crop management of chicory (Compositae) in South Africa.

Chicory, Chicorium intybus L. (Compositae), is a major field crop in the Eastern Cape Province of South Africa. Several pests feed on the leaves of the plant, resulting in reduced yield. The most important of these are the noctuid moths Helicoverpa armigera (Hübner), Chrysodeixis acuta (Walker), and Trichoplusia orichalcea (F.). The use of attract-and-kill traps offers an alternative to broad-based insecticides in the control of these species. Three fields were treated with normal insecticides and three fields with yellow-baited traps. Eight additional traps were placed in each field, with half of the traps containing the insecticide 2,2-dichlorovinyl dimethyl phosphate (dichlorvos) and half without dichlorvos; and half yellow and half green. Total moth numbers and nonphytophage diversity were measured from these eight traps. Although no differences in H. armigera or T. orichalcea catches were observed between insecticide- and trap-treated fields, numbers of C. acuta and the total number of moths were significantly higher in insecticide-treated fields. Yellow traps containing dichlorvos contained more moths than yellow traps without dichlorvos, or green traps with dichlorvos, or green traps without dichlorvos; but they also contained more nonphytophagous insects. Yellow traps also enhanced the catches of thrips on card traps associated with them. These results offer an opportunity for the South African chicory industry to reduce pesticide applications and thus mitigate environmental impacts.

Sperm morphology in four species of African platypleurine cicadas (Hemiptera: Cicadomorpha: Cicadidae).

Mature spermatozoa from four species of platypleurine cicadas (Albanycada albigera, Azanicada zuluensis, Platypleura capensis and P. hirtipennis) were examined by light and electron microscopy. The filiform sperm have a similar ultrastructure in all species but notable variations were found in sperm dimensions. All species produce more than one discrete length of nucleated, motile sperm, a form of polymorphism termed polymegaly. Polymegaly is expressed in two ways: sperm have bi- or trimodal head and tail lengths. The anterior parts of sperm heads are embedded in an elongate homogenous matrix forming a spermatodesm. The conical acrosome is deeply invaginated posteriorly, and sits on top of the nucleus. The acrosomal contents are differentiated internally with an electron-lucent central medulla and a denser cortex. The homogenously electron-dense nucleus is pointed anteriorly and is generally cylindrical, although posteriorly there is a lateral invagination that extends part-way along the nucleus. This invagination houses fine granular material of the centriolar adjunct. Vesicle-like elements that are associated with both the posterior nucleus and the centriolar adjunct are also found within the invagination. Immediately posterior of and adjoining the centriolar adjunct is a pair of mitochondrial derivatives that are elongated and extend for almost the entire length of the tail. The absence of accessory bodies in cicada sperm suggests that within the Cicadomorpha, the families Cicadidae and Cercopidae are closely affiliated.

Human myiasis in rural South Africa is under-reported.

BACKGROUND: Myiasis is the infestation of live tissue of humans and other vertebrates by larvae of flies. Worldwide, myiasis of humans is seldom reported, although the trend is gradually changing in some countries. Reports of human myiasis in Africa are few. Several cases of myiasis were recently seen at the Mthatha Hospital Complex, Mthatha, Eastern Cape Province, South Africa (SA). OBJECTIVE: Because of a paucity of literature on myiasis from this region, surgeons and scientists from Walter Sisulu University, Mthatha, decided to document myiasis cases presenting either at Nelson Mandela Academic Hospital or Umtata General Hospital from May 2009 to April 2013. The objective was to determine the incidence, epidemiology, patient age group and gender, and fly species involved. The effect of season on incidence was also investigated. RESULTS: Twenty-five cases (14 men and 11 women) were recorded in the 4-year study period. The fly species involved were Lucilia sericata, L. cuprina, Chrysomya megacephala, C. chloropyga and Sarcophaga (Liosarcophaga) nodosa, the latter being confirmed as an agent for human myiasis for the first time. The patients were 3 - 78 years old (median 56). Cases were most numerous during spring and summer, and were associated with underlying pathologies typical of ageing. CONCLUSION: Myiasis is a more common medical condition than expected in the Mthatha region. The study shows that human myiasis is still frequently encountered in SA, and there is a need to understand its epidemiology better.

Preliminary observations on the effects of hydrocortisone and sodium methohexital on development of Sarcophaga (Curranea) tibialis Macquart (Diptera: Sarcophagidae), and implications for estimating post mortem interval.

Larvae of Sarcophaga (Curranea) tibialis (S. tibialis) were reared at constant temperature on chicken liver treated with a steroid or a barbiturate at concentrations that would be lethal, half-lethal and twice-lethal doses for humans. Trends to greater mortality at higher drug concentrations were not statistically significant. Larvae exposed to either drug took significantly longer to reach pupation compared to those in the control, while larvae exposed to sodium methohexital passed through pupation significantly faster than those in the control. No systematic relationship was found between drug concentration and development time of larvae or pupae. The total developmental period from hatching to eclosion did not differ between treatments, implying that estimates of post mortem intervals- (PMI) based on the emergence of adult flies will not be affected by the involvement of these drugs in a case. On the other hand, anomalous pupation spans may indicate the presence of barbiturates. These findings are compared with patterns found in another fly fed other contaminants.

A global study of forensically significant calliphorids: implications for identification.

A proliferation of molecular studies of the forensically significant Calliphoridae in the last decade has seen molecule-based identification of immature and damaged specimens become a routine complement to traditional morphological identification as a preliminary to the accurate estimation of post-mortem intervals (PMI), which depends on the use of species-specific developmental data. Published molecular studies have tended to focus on generating data for geographically localised communities of species of importance, which has limited the consideration of intraspecific variation in species of global distribution. This study used phylogenetic analysis to assess the species status of 27 forensically important calliphorid species based on 1167 base pairs of the COI gene of 119 specimens from 22 countries, and confirmed the utility of the COI gene in identifying most species. The species Lucilia cuprina, Chrysomya megacephala, Ch. saffranea, Ch. albifrontalis and Calliphora stygia were unable to be monophyletically resolved based on these data. Identification of phylogenetically young species will require a faster-evolving molecular marker, but most species could be unambiguously characterised by sampling relatively few conspecific individuals if they were from distant localities. Intraspecific geographical variation was observed within Ch. rufifacies and L. cuprina, and is discussed with reference to unrecognised species.

Patterns and processes underlying evolutionary significant units in the Platypleura stridula L. species complex (Hemiptera: Cicadidae) in the Cape Floristic Region, South Africa.

Cicadas have been shown to be useful organisms for examining the effects of distribution, plant association and geographical barriers on gene flow between populations. The cicadas of the Platypleura stridula species complex are restricted to the biologically diverse Cape Floristic Region (CFR) of South Africa. They are thus an excellent study group for elucidating the mechanisms by which hemipteran diversity is generated and maintained in the CFR. Phylogeographical analysis of this species complex using mitochondrial DNA Cytochrome Oxidase I (COI) and ribosomal 16S sequence data, coupled with preliminary morphological and acoustic data, resolves six clades, each of which has specific host-plant associations and distinct geographical ranges. The phylogeographical structure implies simultaneous or near-simultaneous radiation events, coupled with shifts in host-plant associations. When calibrated using published COI and 16S substitution rates typical for related insects, these lineages date back to the late Pliocene - early Pleistocene, coincident with vegetation change, altered drainage patterns and accelerated erosion in response to neotectonic crustal uplift and cyclic Pleistocene climate change, and glaciation-associated changes in climate and sea level.

Recent African derivation of Chrysomya putoria from C. chloropyga and mitochondrial DNA paraphyly of cytochrome oxidase subunit one in blowflies of forensic importance.

Chrysomya chloropyga (Wiedemann) and C. putoria (Wiedemann) (Diptera: Calliphoridae) are closely related Afrotropical blowflies that breed in carrion and latrines, reaching high density in association with humans and spreading to other continents. In some cases of human death, Chyrsomya specimens provide forensic clues. Because the immature stages of such flies are often difficult to identify taxonomically, it is useful to develop DNA-based tests for specimen identification. Therefore we attempted to distinguish between C. chloropyga and C. putoria using mitochondrial DNA (mtDNA) sequence data from a 593-bp region of the gene for cytochrome oxidase subunit one (COI). Twelve specimens from each species yielded a total of five haplotypes, none being unique to C. putoria. Therefore it was not possible to distinguish between the two species using this locus. Maximum parsimony analysis indicated paraphyletic C. chloropyga mtDNA with C. putoria nested therein. Based on these and previously published data, we infer that C. putoria diverged very recently from C. chloropyga.

Molecular identification of some forensically important blowflies of southern Africa and Australia.

One major aspect of research in forensic entomology is the investigation of molecular techniques for the accurate identification of insects. Studies to date have addressed the corpse fauna of many geographical regions, but generally neglected the southern African calliphorid species. In this study, forensically significant calliphorids from South Africa, Swaziland, Botswana and Zimbabwe and Australia were sequenced over an 1167 base pair region of the COI gene. Phylogenetic analysis was performed to examine the ability of the region to resolve species identities and taxonomic relationships between species. Analyses by neighbour-joining, maximum parsimony and maximum likelihood methods all showed the potential of this region to provide the necessary species-level identifications for application to post-mortem interval (PMI) estimation; however, higher level taxonomic relationships did vary according to method of analysis. Intraspecific variation was also considered in relation to determining suitable maximum levels of variation to be expected during analysis. Individuals of some species in the study represented populations from both South Africa and the east coast of Australia, yet maximum intraspecific variation over this gene region was calculated at 0.8%, with minimum interspecific variation at 3%, indicating distinct ranges of variation to be expected at intra- and interspecific levels. This region therefore appears to provide southern African forensic entomologists with a new technique for providing accurate identification for application to estimation of PMI.