Identifying Insects with Incomplete DNA Barcode Libraries, African Fruit Flies (Diptera: Tephritidae) as a Test Case

We propose a general working strategy to deal with incomplete reference libraries in the DNA barcoding identification of species. Considering that (1) queries with a large genetic distance with their best DNA barcode match are more likely to be misidentified and (2) imposing a distance threshold profitably reduces identification errors, we modelled relationships between identification performances and distance thresholds in four DNA barcode libraries of Diptera (n = 4270), Lepidoptera (n = 7577), Hymenoptera (n = 2067) and Tephritidae (n = 602 DNA barcodes). In all cases, more restrictive distance thresholds produced a gradual increase in the proportion of true negatives, a gradual decrease of false positives and more abrupt variations in the proportions of true positives and false negatives. More restrictive distance thresholds improved precision, yet negatively affected accuracy due to the higher proportions of queries discarded (viz. having a distance query-best match above the threshold). Using a simple linear regression we calculated an ad hoc distance threshold for the tephritid library producing an estimated relative identification error <0.05. According to the expectations, when we used this threshold for the identification of 188 independently collected tephritids, less than 5% of queries with a distance query-best match below the threshold were misidentified. Ad hoc thresholds can be calculated for each particular reference library of DNA barcodes and should be used as cut-off mark defining whether we can proceed identifying the query with a known estimated error probability (e.g. 5%) or whether we should discard the query and consider alternative/complementary identification methods

Exploring patterns of cytonuclear incompatibility in Pelargonium section Ciconium

In this thesis I studied cause and effect of Cyto Nuclear Incompatibility (CNI) induced speciation in plants. By combining comparative genomics, phylogenetics and bioinformatics with large scale crossing experiments I was able to infer part of the genetic machinery underlying CNI, determine plastid inheritance, study plastome evolution in detail and establish a possible mechanism for explaining chlorosis.After introducing (Chapter 1) the phenomenon of CNI, its consequences for plant evolution and speciation, and Pelargonium section Ciconium as a model system, I investigate patterns of CNI in a comprehensive series of P. sect. Ciconium (Geraniaceae) interspecific crosses with a single parental population of P. x hortorum in Chapter two. I deduce that one or more nuclear genomic alleles are involved in the expression and management of chloroplasts, and that these act throughout the different life stages. It appears to be the case that the number of alleles needed to explain the observed segregation patters increases with phylogenetic distance between the parent species. Finally, I established that biparental inheritance of chloroplasts is widespread and common across P. sect. Ciconium. All species have the potential to transmit their plastids either paternally or maternally.In Chapter three I use the variation on abundancies of the repetitive part of the genome (the non-coding DNA collectively known as the ‘repeatome’) as a proxy for the nuclear genotype in our crossing experiments (Chapters two and five). In addition, I use genomic repeat abundancy and repeat sequence similarity to reconstruct phylogenetic trees. Hereby the repeat clusters are characters, and their abundancies states. The interpretation of abundancy variation must be done with care, because autopolyploids can cause ‘false’ sister species relationships to occur in the reconstructed tree, based on their genomic sizes, not on shared history. Nevertheless, when I included sequence read variation in the analysis as well, it became clear that the ‘Core-Ciconium’ species share >20 large repeat clusters when compared to P. elongatum and that the species P. multibracteatum, P. quinquelobatum and P. yemenense sp. nov., occurring outside the Cape Floristic Region (CFR), again share several large synapomorphic repeats.In Chapter four, I use a near complete selection of species from P. sect. Ciconium to study plastome evolution in detail. I create a detailed picture of the variation that occurs in P. sect. Ciconium plastomes and present evidence that rrn genes (especially rrn23), encoding ribosomal RNA, and ribosomal proteins have undergone concerted evolution. Rrn23 displays more variation within section P. sect. Ciconium than it does between angiosperm orders. I speculate that one of the consequences of plastome evolution in P. sect. Ciconium (and presumably Pelargonium as well) is the presence of a, structurally, altered plastidial ribosome. In this Chapter I further describe that two of the three available plastid encoded polymerase (PEP) subunits (rpoB and rpoC1) are under positive selection in Ciconium suggesting strong involvement in plastome evolution.Chapter five draws, in part, on the results reported in the previous Chapters. I continued to determine inheritance of plastids, but report newly developed markers for the mitochondria as well. I confirmed the findings from Chapter two that plastids inherit via both parental lines, but biparental inheritance of mitochondria turned out to be rarer. In this Chapter I present three more interspecific crossing series and describe more chlorosis patterns and their associated plastid types. I investigate the occurrence of chlorosis from the chloroplast perspective and find that changes in physico-chemical properties of resulting rpoB peptides occurring in P. sect. Ciconium, by and large, correlate with the observed patterns of chlorosis in F1 interspecific offspring. There is a strong indication that the Ciconium-unique sequence insertions in rpo genes encode parts of the polymerase that are in direct contact with template DNA during transcription. If so, then rpo variation, or PEP structure variation, must be considered, alongside nuclear genomic changes reported in other studies, when understanding plastome evolution in Geraniaceae.In chapter six I discuss the results from the previous chapters in the context of the Bateson-Dobzhansky-Müller model of speciation. I propose a refinement of the model, in which different kinds of selective pressures act at different moments in time, for Pelargonium. I discuss that P. section Ciconium is, likely in an evolutionary phase where CNI is the driver for speciation

Short NoteRange extension of the Lufira Masked Weaver Ploceus ruweti, endemic to Katanga province, Democratic Republic of Congo

No Abstract.OSTRICH 2011, 82(1): 77–7

Is ‘everything everywhere’? Unprecedented cryptic diversity in the cosmopolitan flatworm Gyratrix hermaphroditus

Many nominal species of microscopic animals traditionally fitting the ‘everything is everywhere’ paradigm have been revealed to be complexes of cryptic species. Here, we explore species diversity within the micrometazoan flatworm Gyratrix hermaphroditus—unique among meiofauna because of its global occurrence in a wide variety of brackish, freshwater and marine environments. With maximum likelihood and Bayesian approaches, we analysed 18S, 28S, 5.8S and ITS2 rDNA sequences from 401 specimens across the global distribution of G. hermaphroditus. Generalized Mixed Yule Coalescent (GMYC) and Automatic Barcode Gap Discovery (ABGD) methods delineated 78 and 62 putative species, respectively. This renders G. hermaphroditus one of the most species-rich complexes known to date. Based on shape variations of the male copulatory organ, 14 morphotypes corresponding with molecular clades were identified within the species complex. Within morphotypes, morphometric measurements were able to further discriminate between GMYC species using discriminant analyses. While most putative species occur on local or regional scales, over 10% are distributed over vast distances (>500 km apart) and two GMYC and six ABGD species have colonised multiple continents. This suggests that the cosmopolitanism of the G. hermaphroditus species complex is not just caused by mixing cryptic species with a more limited geographic distribution, but is due to the presence of previously unrecognised cosmopolitan taxa. The wide variation in distribution patterns between putative species indicates that meiofaunal biogeography should not be simplified into ‘everything is everywhere’, but rather entails every ecological state, extending from local endemism to true cosmopolitanism

Adhoc : an R package to calculate ad hoc distance thresholds for DNA barcoding identification

Abstract. Identification by DNA barcoding is more likely to be erroneous when it is based on a large distance between the query (the barcode sequence of the specimen to identify) and its best match in a reference barcode library. The number of such false positive identifications can be decreased by setting a distance threshold above which identification has to be rejected. To this end, we proposed recently to use an ad hoc distance threshold producing identifications with an estimated relative error probability that can be fixed by the user (e.g. 5%). Here we introduce two R functions that automate the calculation of ad hoc distance thresholds for reference libraries of DNA barcodes. The scripts of both functions, a user manual and an example file are available on the JEMU website (http://jemu.myspecies.info/computer-programs) as well as on the comprehensive R archive network (CRAN, http://cran.r-project.org)

a, b, c: Tephritid Best Close Match (BCM) identification.

<p>Proportions of True Positives (TP), False Positives (FP), True Negatives (TN) and False Negatives (FN) at 30 distance thresholds ranging from K2P = 0.165 to K2P = 0.000. For each distance threshold percentages of queries discarded, accuracy ((TP+TN)/total number of queries) and precision (TP/number of not discarded queries) were calculated.</p

a, b, c: Relationships between ID errors and taxon coverage of libraries.

<p>Overall ID errors ((FP+FN)/total number of queries) and relative ID errors (FP/number of not discarded queries) at 30 arbitrary BCM distance thresholds in a) Diptera, b) Lepidoptera and c) Hymenoptera. In the 100% taxon coverage simulation, each query had at least a conspecific in the reference database. In the remaining simulations 25%, 50% and 75% of query species were not represented in the reference library (corresponding to 75%, 50% and 25% of taxon coverage). Simulations of 75%, 50% and 25% taxon coverage were repeated three times (standard errors as error bars). For each simulation, <i>ad hoc</i> distance thresholds (THR_{K2P_0.05}) corresponding to a relative ID error <0.05 were inferred from linear fitting.</p

The mitochondrial cytochrome <i>c</i> oxidase I gene reveals phylogeographic structure in the African Goshawk <i>Accipiter tachiro</i> (Accipitridae)

<div><p>We used a 298 bp fragment of the mitochondrial cytochrome <i>c</i> oxidase subunit I gene (COI) to examine sequence variation in (mostly) museum specimens of the African Goshawk <i>Accipiter tachiro</i>. Our results showed two clades with high bootstrap support in a phylogenetic analysis and two groups in a nonmetric multidimensional scaling (NMDS) analysis. Each of the two phylogenetic clades corresponded to one of the NMDS groups. One clade comprised haplotypes of the subspecies <i>A. t. lopezi</i>, <i>A. t. macroscelides</i>, <i>A. t. toussenelli</i> and <i>A. t. canescens</i> and corresponded to the morphospecies <i>A. toussenelii</i>. This taxon has a more north-western distribution. The second clade comprised haplotypes of the subspecies <i>A. t. sparsimfasciatus</i>, <i>A. t. pembaensis</i> and <i>A. t. tachiro</i> and corresponded to the morphospecies <i>A. tachiro</i>, which has a more south-eastern distribution. Furthermore, one branch corresponded to the morphospecies <i>A. t. unduliventer</i>, which is confined to the Ethiopian highlands. The genetic divergence observed among the three <i>A. tachiro</i> morphospecies appeared concordant with the ecological and morphological divergence and suggests the existence of three putative species. Within <i>A. tachiro</i> and <i>A. toussenelii</i> there is substantial morphological, but very little genetic, differentiation among subspecies.</p></div