Penny J. Gullan

Secondary (γ-Proteobacteria) Endosymbionts Infect the Primary (β-Proteobacteria) Endosymbionts of Mealybugs Multiple Times and Coevolve with Their Hosts

ABSTRACT Mealybugs (Hemiptera, Coccoidea, Pseudococcidae) are plant sap-sucking insects that have within their body cavities specialized cells containing prokaryotic primary endosymbionts (P-endosymbionts). The P-endosymbionts have the unusual property of containing within their cytoplasm prokaryotic secondary endosymbionts (S-endosymbionts) [C. D. von Dohlen, S. Kohler, S. T. Alsop, and W. R. McManus, Nature (London) 412:433-436, 2001]. Four-kilobase fragments containing 16S-23S ribosomal DNA (rDNA) were obtained from the P-endosymbionts of 22 mealybug species and the S-endosymbionts of 12 representative species. Phylogenetic analyses of the P-endosymbionts indicated that they have a monophyletic origin and are members of the β-subdivision of the Proteobacteria; these organisms were subdivided into five different clusters. The S-endosymbionts were members of the γ-subdivision of the Proteobacteria and were grouped into clusters similar to those observed with the P-endosymbionts. The S-endosymbiont clusters were distinct from each other and from other insect-associated bacteria. The similarity of the clusters formed by the P- and S-endosymbionts suggests that the P-endosymbionts of mealybugs were infected multiple times with different precursors of the S-endosymbionts and once the association was established, the P- and S-endosymbionts were transmitted together. The lineage consisting of the P-endosymbionts of mealybugs was given the designation “Candidatus Tremblaya” gen. nov., with a single species, “Candidatus Tremblaya princeps” sp. nov. The results of phylogenetic analyses of mitochondrial DNA fragments encoding cytochrome oxidase subunits I and II from four representative mealybug species were in agreement with the results of 16S-23S rDNA analyses, suggesting that relationships among strains of “Candidatus T. princeps” are useful in inferring the phylogeny of their mealybug hosts.

Phylogenetic evidence for two new insect-associated Chlamydia of the family Simkaniaceae.

On the basis of 16S–23S ribosomal DNA analyses, the whitefly Bemisia tabaci (Sternorrhyncha, Aleyrodidae) and the eriococcid Eriococcus spurius (Sternorrhyncha, Eriococcidae) were each found to harbor novel related chlamydial species within the family Simkaniaceae. The generic designation Fritschea gen. nov. is proposed to accommodate the two species, F. bemisiae sp. nov. and F. eriococci sp. nov. The finding of chlamydial 16S–23S ribosomal DNA in B. tabaci is consistent with a previous electron microscopy study which found that bacteriocytes of this species contain structures that we consider to resemble the elementary and reticulate bodies of chlamydia (Costa HS, Westcot DM, Ullman DE, Rosell R, Brown JK, Johnson MW. Protoplasma 189:194–202, 1995). The cloning and sequencing of a 16.6 kilobase DNA fragment from F. bemisiae indicated that it contains six genes encoding for proteins similar to those found in other species of chlamydia. These results extend the range of organisms that harbor chlamydia.

Phylogenetic analysis of mealybugs (Hemiptera: Coccoidea: Pseudococcidae) based on DNA sequences from three nuclear genes, and a review of the higher classification.

Abstract. Mealybugs (Hemiptera: Pseudococcidae) are small, plant‐sucking insects which comprise the second largest family of scale insects (Coccoidea). Relationships among many pseudococcid genera are poorly known and there is no stable higher level classification. Here we review previous hypotheses on relationships and classification and present the first comprehensive phylogenetic study of the Pseudococcidae based on analysis of nucleotide sequence data. We used three nuclear genes, comprising two noncontiguous fragments of elongation factor 1α (EF‐1α 5′ and EF‐1α 3′), fragments of the D2 and D10 expansion regions of the large subunit ribosomal DNA gene (28S), and a region of the small subunit ribosomal DNA gene (18S). We sampled sixty‐four species of mealybug belonging to thirty‐five genera and representing each of the five subfamilies which had been recognized previously, and included four species of Puto (Putoidae) and one species each of Aclerda (Aclerdidae) and Icerya (Margarodidae), using Icerya as the most distant outgroup. A combined analysis of all data found three major clades of mealybugs which we equate to the subfamilies Pseudococcinae, Phenacoccinae and Rhizoecinae. Within Pseudococcinae, we recognize the tribes Pseudococcini (for Pseudococcus, Dysmicoccus, Trionymus and a few smaller genera), Planococcini (for Planococcus and possibly Planococcoides) and Trabutinini (represented by a diverse range of genera, including Amonostherium, Antonina, Balanococcus, Nipaecoccus and non‐African Paracoccus), as well as the Ferrisia group (for Ferrisia and Anisococcus), some ungrouped African taxa (Grewiacoccus, Paracoccus, Paraputo and Vryburgia), Chaetococcus bambusae and Maconellicoccus. The ‘legless’ mealybugs Antonina and Chaetococcus were not closely related and thus we confirmed that the Sphaerococcinae as presently constituted is polyphyletic. In our analyses, the subfamily Phenacoccinae was represented by just Phenacoccus and Heliococcus. The hypogeic mealybugs of the Rhizoecinae usually formed a monophyletic group sister to all other taxa. Our molecular data also suggest that the genera Pseudococcus, Dysmicoccus, Nipaecoccus and Paracoccus are not monophyletic (probably polyphyletic) and that Phenacoccus may be paraphyletic, but further sampling of species and genes is required. We compare our phylogenetic results with published information on the intracellular endosymbionts of mealybugs and hypothesize that the subfamily Pseudococcinae may be characterized by the possession of β‐Proteobacteria (primary endosymbionts) capable of intracellular symbiosis with γ‐Proteobacteria (secondary endosymbionts). Furthermore, our data suggest that the identities of the secondary endosymbionts may be useful in inferring mealybug relationships. Finally, cloning polymerase chain reaction products showed that paralogous copies of EF‐1α were present in at least three taxa. Unlike the situation in Apis and Drosophila, the paralogues could not be distinguished by either the presence/absence or position of an intron.

A subfamily-level classification of mealybugs (Hemiptera: Pseudococcidae) based on integrated molecular and morphological data

Abstract The mealybugs (Hemiptera: Coccoidea: Pseudococcidae) are a speciose and ubiquitous group of sap‐sucking plant parasites, many of which are very serious agricultural pests. There has been much work on the alpha‐level taxonomy, amounting to the description of more than 2000 species, but suprageneric relationships remain poorly known. Downie & Gullan reviewed proposed schemes for a mealybug subfamily‐level classification and used DNA sequence data from three nuclear genes to infer the mealybug phylogeny. They recognized three subfamilies: the Rhizoecinae, the Phenacoccinae, and the Pseudococcinae; and within the Pseudococcinae, recognized the tribes Pseudococcini, Trabutinini, and Planococcini. Excepting the Trabutinini, none of these groupings was well‐supported. We improve our estimation of the mealybug phylogeny by: (i) increasing the taxon sampling by 50%; (ii) adding a morphological character matrix; and (iii) performing mixed model Bayesian and maximum likelihood inference procedures. We recover two primary clades within the Pseudococcidae, to which we apply the subfamily names Phenacoccinae and Pseudococcinae. Within the Phenacoccinae, we recover support for the monophyly of the hypogaeic mealybugs (Rhizoecini), and within the Pseudococcinae, we find support for the tribes Pseudococcini, Trabutinini, and Planococcini. Our results suggest a clear sequence of (i) β‐Proteobacteria primary endosymbionts infecting the mycetome of the most recent common ancestor of the Pseudococcinae, followed by (ii) γ‐Proteobacteria secondary endosymbionts infecting the primary β‐Proteobacteria endosymbionts. For each subfamily, we provide a morphological diagnosis and a comprehensive list of included genera. We resurrect the genus Ceroputo Šulc, currently under synonymy with the genus Puto Signoret (Putoidae), and place it in the mealybug subfamily Phenacoccinae. Greenoripersia kaiseri Bodenheimer is transferred from the Pseudococcidae to the Eriococcidae.

Sternorrhyncha: (Jumping Plant-Lice, Whiteflies, Aphids, and Scale Insects)

Publisher Summary This chapter focuses on the sternorrhyncha, suborder of the order Hemiptera. It comprising some 16,000 described species and contains four major groups, all entirely phytophagous, and usually recognized as superfamilies: the Psylloidea (psylloids or jumping plant-lice); Aleyrodoidea (whitefl ies); Aphidoidea (aphids or aphidoids); and Coccoidea (scale insects or coccoids). Insects belonging to the Hemiptera are unique in having their mouthparts forming a rostrum that comprises mandibles and maxillae modified as needle- or thread-like stylets lying within a grooved labium. Two pairs of stylets interlock to form two canals, one delivering saliva and the other uptaking plant or animal fluid. Sternorrhynchans use their stylets to probe plant tissues intracellularly or intercellularly. The tips of the stylets always enter cells at the site of ingestion, which is often phloem-sieve element. Generally, stylet penetration is accompanied by secretion of solidifying saliva that forms a sheath around the stylets. Other hemipterans mostly probe intracellularly, may or may not secrete salivary sheaths, and ingest from a wider variety of plant or animal tissues. Most sternorrhynchans are phloem feeders, and thus have a diet rich in carbohydrates and deficient in amino acids and other nitrogenous compounds. Generally, there is an intimate association with intracellular bacteria, called endosymbionts, which are housed in special tissue, bacteriomes or mycetomes, and contribute nutrition to the insect host.

Trans-Tasman Platycoelostoma Morrison (Hemiptera: Coccoidea: Margarodidae) on endemic Cupressaceae, and the phylogenetic history of margarodids

A new species of Platycoelostoma Morrison (Hemiptera: Coccoidea: Margarodidae) is described from the Tasmanian endemic conifer, Diselma archeri (Cupressaceae), growing in alpine heathland. Its sister species, P. compressa (Maskell), occurs in alpine and subalpine New Zealand on Libocedrus bidwilli (Cupressaceae). Vicariance dating provides a minimum age for Platycoelostoma of about 80 Ma. Cladistic analysis of morphological data from adult females and first‐instar nymphs suggests that Platycoelostoma is the sister genus of Callipappus Guérin‐Méneville and also is more closely related to members of subfamilies Margarodinae and Xylococcinae than to the New Zealand and South American genera of Coelostomidiinae, to which Platycoelostoma traditionally has been linked. The analysis also indicates that most traditional tribal groupings within Margarodidae are monophyletic, but that three (Coelostomidiinae, Margarodinae and Xylococcinae) of the five subfamilies in the widely used higher classification of Morrison may be either paraphyletic or polyphyletic as currently defined. Only Monophlebinae and Steingeliinae have adequate support from morphological characters. Relationships among subfamilies and many tribes are unresolved. These results lend some support to the classification previously proposed by Koteja, in which all margarodid subfamilies and a few tribes are elevated to the rank of family. Furthermore, the analysis suggests that Marchalini and Monophlebinae (in which all immature female instars have well developed appendages) are derived with respect to other margarodids and thus development via legless intermediate female instars (‘cyst’ form) may be plesiomorphic within margarodids. This hypothesis is weakly supported and requires corroboration from independent data. Platycoelostoma is transferred to tribe Callipappini, which is redefined, but the subfamily placement of this tribe must remain uncertain until the higher classification and rank of the various margarodid taxa can be re‐evaluated. A revised generic description of Platycoelostoma, based on the adult and intermediate female instars and the first instar of both species, and a key to separate the female instars of both species are provided. The first‐instar nymph, all female instars and the third‐instar male (the prepupa) of Platycoelostoma tasmanicum sp.n. are described and illustrated. Scanning electron micrographs depict the unusual cuticular features of the third‐instar female.

Phylogeny of Insects

Publisher Summary This chapter discusses the different sources of evidence for the phylogenies. Well-founded and less well-founded traditional, even refuted, relationships are discussed, and if resolution appears to be lacking, this inadequacy is identified. Insects belong to arguably the most successful major lineage of the phylum Arthropoda, the joint-legged animals. The latter clade comprises myriapods (centipedes, millipedes, and their relatives), chelicerates (horseshoe crabs and arachnids), crustaceans (crabs, shrimps, and relatives), and hexapods (the six-legged arthropods, Insecta, and their relatives). Lobopods (onychophorans) sometimes have been included, but now almost universally are considered to lie outside of Arthropoda. Although traditionally each major arthropod group has been considered monophyletic, most have been suspected of non-monophyly by at least a few investigators. Results of molecular analyses have provided frequent challenges, particularly in suggesting the possible paraphyly of myriapods and of crustaceans. Ideas concerning the phylogenetic relationships among the major taxa of arthropods, and the included insects, are dynamic. Although there is a single evolutionary history, efforts to uncover this phylogeny vary between different researchers, techniques, and character systems studied. No technique or character system alone can guarantee to reveal the true relationships of the studied taxa; in actuality, convergent (homoplastic) similarity that confuses relationships is common to all data. The evidence behind traditional systems, representing perhaps the thorough understanding of a single character system rather than an integration of all knowledge, sometimes cannot withstand detailed scrutiny. Molecular sequence data often appear to overturn previous ideas derived from morphological interpretation but may be misleading due to undersampling, unrecognized sampling of alternative gene duplicates (paralogs), and/or inappropriate analyses.

Molecular data reveal convergent reproductive strategies in iceryine scale insects (Hemiptera: Coccoidea: Monophlebidae), allowing the re‐interpretation of morphology and a revised generic classification

Abstract The scale insect tribe Iceryini (Coccoidea: Monophlebidae) is a group of relatively large and polyphagous insects found worldwide. Currently, the tribe contains about 80 named species placed in seven genera, which are diagnosed largely on features associated with egg protection. We reconstruct the phylogeny of the Iceryini on the basis of nucleotide sequence data from nuclear ribosomal (18S and D2, D3 and D10 regions of 28S) and protein‐coding (histone H3) gene regions of 40 iceryine species representing six of the seven genera and seven outgroup taxa, mostly from two other tribes of Monophlebidae. Bayesian and maximum parsimony analyses recover a monophyletic tribe and clades that correspond more to geography than to the existing morphology‐based classification. Gueriniella Fernald is sister to the rest of the Iceryini and the genera Crypticerya Cockerell, Icerya Signoret and Steatococcus Ferris are not monophyletic. Our data imply that the distinctive iceryine reproductive strategies, such as protecting eggs in a waxy ovisac or inside a marsupium, are poor indicators of relationships. On the basis of molecular relationships and the re‐examination of morphological characters, we recognize only five genera of Iceryini –Crypticerya, Echinicerya Morrison, Gigantococcus Pesson & Bielenin, Gueriniella and Icerya – and substantially revise the generic concepts of Crypticerya, Gigantococcus and Icerya. We provide a key to the genera based on adult females. We redescribe and illustrate the adult female and first‐instar nymph of the type species Crypticerya rosae (Riley & Howard), Echinicerya anomala Morrison, Gigantococcus maximus (Newstead) (adult female only), Gueriniella serratulae (Fabricius) and Icerya seychellarum (Westwood). We recognize Auloicerya Morrison as a junior synonym (syn.n.) of Icerya, and transfer the two Auloicerya species to Icerya as I. acaciae (Morrison & Morrison) comb.n. and I. australis Maskell comb.rev. We recognize Steatococcus and Proticerya Cockerell as junior synonyms (syn.n.) of Crypticerya. From Steatococcus, we transfer five species to Crypticerya [C. mexicana Cockerell & Parrott comb.rev., C. morrilli (Cockerell) comb.n., C. tabernicola (Ferris) comb.n., C. townsendi Cockerell comb.rev., C. tuberculata (Morrison) comb.n.], four species to Gigantococcus [Gi. euphorbiae (Brain) comb.n., Gi. gowdeyi (Newstead) comb.n., Gi. madagascariensis (Mamet) comb.n., Gi. theobromae (Newstead) comb.n.] and three species to Icerya [I. assamensis (Rao) comb.n., I nudata Maskell comb.rev., I. samaraia (Morrison) comb.n.]. From Icerya, we transfer 14 species to Crypticerya [C. brasiliensis (Hempel) comb.n., C. colimensis (Cockerell) comb.n., C. flava (Hempel) comb.n., C. flocculosa (Hempel) comb.n., C. genistae (Hempel) comb.n., C. littoralis (Cockerell) comb.n., C. luederwaldti (Hempel) comb.n., C. minima (Morrison) comb.n., C. montserratensis (Riley & Howard) comb.n., C. palmeri (Riley & Howard) comb.n., C. rileyi (Cockerell) comb.n., C. similis (Morrison) comb.n., C. subandina (Leonardi) comb.n., C. zeteki (Cockerell) comb.n.] and nine species to Gigantococcus [Gi. alboluteus (Cockerell) comb.n., Gi. bimaculatus (De Lotto) comb.n., Gi. brachystegiae (Hall) comb.n., Gi. longisetosus (Newstead) comb.n., Gi. nigroareolatus (Newstead) comb.n., Gi. pattersoni (Newstead) comb.n., Gi. schoutedeni (Vayssière) comb.n., Gi. splendidus (Lindinger) comb.n., Gi. sulfureus (Lindinger) comb.n.]. From Crypticerya, we transfer seven species to Icerya [I. clauseni (Rao) comb.n., I. jacobsoni Green comb.rev., I. jaihind (Rao) comb.n., I. kumari (Rao) comb.n., I. mangiferae (Tang & Hao) comb.n., I. natalensis (Douglas) comb.rev., I. nuda Green comb.rev.] and five species to Gigantococcus [Gi. bicolor (Newstead) comb.n., Gi. cajani (Newstead) comb.n., Gi. caudatus (Newstead) comb.n., Gi. ewarti (Newstead) comb.n., Gi. rodriguesi (Castel‐Branco) comb.n.]. Both I. hyperici (Froggatt) and Palaeococcus dymocki (Froggatt) are syn.n. of I. nudata (all previously placed in Steatococcus). We recognize I. maynei Vayssière as a syn.n. of Gi. nigroareolatus, I. tremae Vayssière as a syn.n. of Gi. schoutedeni and I. townsendi plucheae Cockerell as a syn.n. of C. townsendi. We revalidate the species name I. crocea Green stat.reval. In addition, we transfer I. taunayi Hempel to Laurencella Foldi (Monophlebidae: Llaveiini) as L. taunayi (Hempel) comb.n. Four species, Coccus hirticornis Boyer de Fonscolombe, I. chilensis Hempel, I. insulans Hempel and I. paulista Hempel, are considered incertae sedis. We designate lectotypes for C. rosae, E. anomala and I. candida (a junior synonym of I. seychellarum). Following this revision, we recognize 74 species of Iceryini, distributed as follows: 22 in Crypticerya, one in Echinicerya, 19 in Gigantococcus, two in Gueriniella and 30 in Icerya.

Coccidae, Pseudococcidae, Ortheziidae, and Monophlebidae (Hemiptera: Coccoidea) of Espírito Santo, Brazil

Culik, M.P., Martins, D.S., Ventura, J.A., Peronti, A.L.B.G., Gullan, P.Y. & Kondo, T. Coccidae, Pseudococcidae, Ortheziidae, and Monophlebidae (Hemiptera: Coccoidea) of Espirito Santo, Brazil. Biota Neotrop. Sep/Dez 2007 vol. 7, no. 3 http://www.biotaneotropica.org.br/v7n3/pt/abstract?article+bn00507032007. ISSN 1676-0603. New plant hosts are recorded for nine scale insect species recently collected in Espirito Santo, Brazil, and eleven scale insect species are recorded for the first time from the state: Ceroplastes floridensis Comstock, Coccus longulus (Douglas), Coccus viridis (Green), Eucalymnatus tesselatus (Signoret), Pseudokermes sp., Saissetia coffeae (Walker), Phenacoccus madeirensis Green, Pseudococcus jackbeardsleyi Gimpel & Miller, Pseudococcus longispinus (Targioni Tozzetti), Icerya purchasi Maskell, and Icerya genistae Hempel. This is also the first record of Co. longulus in Brazil. Information on the host plants and geographic distribution of the 26 species of scale insects of the families Coccidae, Pseudococcidae, Ortheziidae, and Monophlebidae, currently known from Espirito Santo is provided.

First records of two mealybug species in Brazil and new potential pests of papaya and coffee

Abstract Five mealybug (Hemiptera: Pseudococcidae) plant pest species: Dysmicoccus grassii (Leonardi), Ferrisia malvastra (McDaniel), Ferrisia virgata (Cockerell), Phenacoccus tucumanus Granara de Willink, and Pseudococcus elisae Borchsenius are recorded for the first time in the state of Espírito Santo, Brazil. These are the first records of D. grassii in Brazil, from papaya (Carica papaya, Caricaceae), and from coffee (Coffea canephora, Rubiaceae). Ferrisia malvastra is also newly recorded in Brazil, where it was found on Bidens pilosa (Asteraceae). Ferrisia virgata was collected from an unidentified weed and Phenacoccus tucumanus from Citrus sp. (Rutaceae). Plotococcus capixaba Kondo was found on pitanga (Eugenia cf. pitanga, Myrtaceae) and Pseudococcus elisae on Coffea canephora, which are new host records for these mealybugs.