Laura M. Boykin

Species delimitation and global biosecurity

Species delimitation directly impacts on global biosecurity. It is a critical element in the decisions made by national governments in regard to the flow of trade and to the biosecurity measures imposed to protect countries from the threat of invasive species. Here we outline a novel approach to species delimitation, “tip to root”, for two highly invasive insect pests, Bemisia tabaci (sweetpotato whitefly) and Lymantria dispar (Asian gypsy moth). Both species are of concern to biosecurity, but illustrate the extremes of phylogenetic resolution that present the most complex delimitation issues for biosecurity; B. tabaci having extremely high intra-specific genetic variability and L. dispar composed of relatively indistinct subspecies. This study tests a series of analytical options to determine their applicability as tools to provide more rigorous species delimitation measures and consequently more defensible species assignments and identification of unknowns for biosecurity. Data from established DNA barcode datasets (COI), which are becoming increasingly considered for adoption in biosecurity, were used here as an example. The analytical approaches included the commonly used Kimura two-parameter (K2P) inter-species distance plus four more stringent measures of taxon distinctiveness, (1) Rosenbergs reciprocal monophyly, (P(AB)), 1 (2) Rodrigos (P(randomly distinct)), 2 (3) genealogical sorting index, (gsi), 3 and (4) General mixed Yule- coalescent (GMYC).4,5 For both insect datasets, a comparative analysis of the methods revealed that the K2P distance method does not capture the same level of species distinctiveness revealed by the other three measures; in B. tabaci there are more distinct groups than previously identified using the K2P distances and for L. dipsar far less variation is apparent within the predefined subspecies. A consensus for the results from P(AB), P(randomly distinct) and gsi offers greater statistical confidence as to where genetic limits might be drawn. In the species cases here, the results clearly indicate that there is a need for more gene sampling to substantiate either the new cohort of species indicated for B. tabaci or to detect the established subspecies taxonomy of L. dispar. Given the ease of use through the Geneious species delimitation plugins, similar analysis of such multi-gene datasets would be easily accommodated. Overall, the tip to root approach described here is recommended where careful consideration of species delimitation is required to support crucial biosecurity decisions based on accurate species identification.

Improved DNA Barcoding Method for Bemisia tabaci and Related Aleyrodidae: Development of Universal and Bemisia tabaci Biotype-Specific Mitochondrial Cytochrome c Oxidase I Polymerase Chain Reaction Primers

ABSTRACT Whiteflies, heteropterans in the family Aleyrodidae, are globally distributed and severe agricultural pests. The mitochondrial cytochrome c oxidase I (mtCOI) sequence has been used extensively in whitefly phylogenetic comparisons and in biotype identification of the agriculturally important Bemisia tabaci (Gennadius) whitefly. Because of the economic importance of several whitefly genera, and the invasive nature of the B and the Q biotypes of Bemisia tabaci, mtCOI sequence data are continually generated from sampled populations worldwide. Routine phylogenetic comparisons and biotype identification is done through amplification and sequencing of an ≈800-bp mtCOI DNA fragment. Despite its routine use, published primers for amplification of this region are often inefficient for some B. tabaci biotypes and especially across whitefly species. Through new sequence generation and comparison to available whitefly mtCOI sequence data, a set of polymerase chain reaction (PCR) amplification primers (Btab-Uni primers) were identified that are more efficient at amplifying ≈748 bp of the ≈800-bp fragment currently used. These universal primers amplify an mtCOI fragment from numerous B. tabaci biotypes and whitefly genera by using a single amplification profile. Furthermore, mtCOI PCR primers specific for the B, Q, and New World biotypes of B. tabaci were designed that allow rapid discrimination among these biotypes. These primers produce a 478-, 405-, and 303-bp mtCOI fragment for the B, New World, and Q biotypes, respectively. By combining these primers and using rapid PCR and electrophoretic techniques, biotype determination can be made within 3 h for up to 96 samples at a time.

A practical guide to identifying members of the Bemisia tabaci species complex: and other morphologically identical species

Members of the Bemisia tabaci species complex (whiteflies) are a considerable threat to a broad range of agriculture and horticulture food and fiber crops. There are hundreds of papers a year published on the members of B. tabaci species complex, many failing to either correctly identify the species involved or confusing identity. Correct identification is a crucial first step in any study, yet all too often, especially in cases where the primary focus of the study is plant pathology, is overlooked. The whitefly research community has struggled for years with common terminology and consistent methods for species identification of various members of the complex due to 1) the lack of a reliable global genetic resource and 2) the complexities of the phylogenetic methods needed to identify unknown individuals correctly. The goal of this paper are to provide a practical guide for identifying unknown whiteflies using a global curated dataset of mitochondrial COI that is freely available at http://dx.doi.org/10.4225/08/50EB54B6F1042. The methods and resources outlined here can be readily extended to other species that are morphologically indistinguishable.

Transmission specificities of plant viruses with the newly identified species of the Bemisia tabaci species complex

Bemisia tabaci has had a colorful nomenclatural past and is now recognized as a species complex. This new species framework has added many new areas of research including adding new insight into the virus transmission specificity of the species in the B. tabaci species complex. There is a wide disparity in what is known about the transmission of plant viruses by different members of the B. tabaci species complex. In this paper, we have synthesized the transmission specificities of the plant viruses transmitted by species belonging to the complex. There are five genera of plant viruses with members that are transmitted by species of the B. tabaci species complex. The transmission of viruses belonging to two of these, Begomovirus and Crinivirus, are well studied and much is known in regards to the relationship between species and transmission and etiology. This is in contrast to viruses of the genera, Torradovirus and Carlavirus, for which very little is known inregards to their transmission. This is the first attempt to integrate viral data within the new B. tabaci species complex framework. It is clear that matching historical transmission data with the current species framework is difficult due to the lack of awareness of the underlying genetic diversity within B. tabaci. We encourage all researchers to determine which species of B. tabaci they are using to facilitate association of phenotypic traits with particular members of the complex.

Is agriculture driving the diversification of the Bemisia tabaci species complex (Hemiptera: Sternorrhyncha: Aleyrodidae)?: Dating, diversification and biogeographic evidence revealed

BackgroundHumans and insect herbivores are competing for the same food crops and have been for thousands of years. Despite considerable advances in crop pest management, losses due to insects remain considerable. The global homogenisation of agriculture has supported the range expansion of numerous insect pests and has been driven in part by human-assisted dispersal supported through rapid global trade and low-cost air passenger transport. One of these pests, is the whitefly, Bemisia tabaci, a cryptic species complex that contains some of the world’s most damaging pests of agriculture. The complex shows considerable genetic diversity and strong phylogeographic relationships. One consequence of the considerable impact that members of the B. tabaci complex have on agriculture, is the view that human activity, particularly in relation to agricultural practices, such as use of insecticides, has driven the diversification found within the species complex. This has been particularly so in the case of two members of the complex, Middle East-Asia Minor 1 (MEAM1) and Mediterranean (MED), which have become globally distributed invasive species. An alternative hypothesis is that diversification is due to paleogeographic and paleoclimatological changes.ResultsThe idea that human activity is driving speciation within the B. tabaci complex has never been tested, but the increased interest in fossil whiteflies and the growth in molecular data have enabled us to apply a relaxed molecular clock and so estimate divergence dates for the major lineages within the B. tabaci species complex. The divergence estimates do not support the view that human activity has been a major driver of diversification.ConclusionsOur analysis suggests that the major lineages within the complex arose approximately 60–30 mya and the highly invasive MED and MEAM1 split from the rest of the species complex around 12 mya well before the evolution of Homo sapiens and agriculture. Furthermore, the divergence dates coincide with a period of global diversification that occurred broadly across the plant and animal kingdoms and was most likely associated with major climatic and tectonic events.

Analyses of Twelve New Whole Genome Sequences of Cassava Brown Streak Viruses and Ugandan Cassava Brown Streak Viruses from East Africa: Diversity, Supercomputing and Evidence for Further Speciation

Cassava brown streak disease is caused by two devastating viruses, Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) which are frequently found infecting cassava, one of sub-Saharan Africa’s most important staple food crops. Each year these viruses cause losses of up to

Bemisia tabaci nomenclature: lessons learned

100 million USD and can leave entire families without their primary food source, for an entire year. Twelve new whole genomes, including seven of CBSV and five of UCBSV were uncovered in this research, doubling the genomic sequences available in the public domain for these viruses. These new sequences disprove the assumption that the viruses are limited by agro-ecological zones, show that current diagnostic primers are insufficient to provide confident diagnosis of these viruses and give rise to the possibility that there may be as many as four distinct species of virus. Utilizing NGS sequencing technologies and proper phylogenetic practices will rapidly increase the solution to sustainable cassava production.

The characteristics and expression profiles of the mitochondrial genome for the Mediterranean species of the Bemisia tabaci complex.

BACKGROUND The nomenclature used within the whitefly research community for different putative species within Bemisia tabaci (sensu Russell) remains highly variable and confused. This was evident by the many different naming schemes researchers were using in their presentations at the 1st International Whitefly Symposium in Kolymbari, Crete, Greece (20-24 May 2013). I wanted to try to document how we, as a community, have arrived at such a state of confused nomenclature. This also included an investigation of the nomenclature used in the literature (from 2002 to 2012) by means of two online search tools (Web of Science and Scirus). RESULTS Nomenclatural data were collected at the 1st International Whitefly Symposium, based on oral presentations and posters. There were 17 different names used for the MED species and 12 different names used for the MEAM1 species of the B. tabaci species complex. Investigation of the literature revealed limited uptake of the intermediate names. CONCLUSION The intermediate names for the various species in the B. tabaci species complex - MED, MEAM1, New World, etc. - are not being used by the wider whitefly community. To move forwarrd as a community, we must work towards a formal revision of the B. tabaci complex.

PPR-SMRs: Ancient proteins with enigmatic functions

BackgroundThe whiteflies under the name Bemisia tabaci (Gennadius) (Aleyrodidae: Hemiptera) are species complex of at least 31 cryptic species some of which are globally invasive agricultural pests. Previously, the mitochondrial genome (mitogenome) of the indigenous New World B. tabaci species was sequenced and major differences of gene order from the postulated whitefly ancestral gene order were found. However, the sequence and gene order of mitogenomes in other B. tabaci species are unknown. In addition, the sequence divergences and gene expression profiles of mitogenomes in the B. tabaci species complex remain completely unexplored.ResultsIn this study, we obtained the complete mitogenome (15,632 bp) of the invasive Mediterranean (MED), which has been identified as the type species of the B. tabaci complex. It encodes 37 genes, including 13 protein-coding genes (PCGs), 2 ribosomal RNAs and 22 transfer RNAs (tRNA). Comparative analyses of the mitogenomes from MED and New World (previously published) species reveal that there are no gene arrangements. Based on the Illumina sequencing data, the gene expression profile of the MED mitogenome was analyzed. We found that a number of genes were polyadenylated and the partial stop codons in cox1, cox2 and nd5 are completed via polyadenylation that changed T to the TAA stop codon. In addition, combining the transcriptome with the sequence alignment data, the possible termination site of some PCGs were defined. Our analyses also revealed that atp6 and atp8, nd4 and nd4l, nd6 and cytb were found on the same cistronic transcripts, whereas the other mature mitochondrial transcripts were monocistronic. Furthermore, RT-PCR analyses of the mitochondrial PCGs expression in different developmental stages revealed that the expression level of individual mitochondrial genes varied in each developmental stage of nymph, pupa and adult. Interestingly, mRNA levels showed significant differences among genes located in the same transcription unit suggesting that mitochondrial mRNA abundance is heavily modulated by post-transcriptional regulation.ConclusionsThis work provides novel insights into the mitogenome evolution of B. tabaci species and demonstrates that utilizing RNA-seq data to obtain the mitogenome and analyze mitochondrial gene expression characteristics is practical.

Integration of complete chloroplast genome sequences with small amplicon datasets improves phylogenetic resolution in Acacia

A small subset of the large pentatricopeptide repeat (PPR) protein family in higher plants contain a C-terminal small MutS-related (SMR) domain. Although few in number, they figure prominently in the chloroplast biogenesis and retrograde signaling literature due to their striking mutant phenotypes. In this review, we summarize current knowledge of PPR-SMR proteins focusing on Arabidopsis and maize proteomic and mutant studies. We also examine their occurrence in other organisms and have determined by phylogenetic analysis that, while they are limited to species that contain chloroplasts, their presence in algae and early branching land plant lineages indicates that the coupling of PPR motifs and an SMR domain into a single protein occurred early in the evolution of the Viridiplantae clade. In addition, we discuss their possible function and have examined conservation between SMR domains from Arabidopsis PPR proteins with those from other species that have been shown to possess endonucleolytic activity.