Evolution of the planthoppers (Insecta: Hemiptera: Fulgoroidea)

The planthopper superfamily Fulgoroidea (Insecta: Hemiptera) comprises approximately 20 described insect families, depending on which classification is followed. Multiple competing hypotheses of fulgoroid phylogeny have been published, based on either morphological character coding or DNA sequence data; however, those hypotheses disagree in several key aspects regarding the evolution of planthoppers. The current paper seeks to test these hypotheses, including the Asche (Asche, M. 1987. Preliminary thoughts on the phylogeny of Fulgoromorpha (Homoptera Auchenorrhyncha). In: Proceedings of the 6th Auchenorrhyncha Meeting, Turin, Italy, 7-11 September, 1987, pp. 47-53.) hypothesis of a trend in ovipositor structure, which may be correlated with planthopper feeding ecology. Presented here are phylogenetic reconstructions of Fulgoroidea based on analysis of DNA nucleotide sequence data from four loci (18S rDNA, 28S rDNA, Histone 3, and Wingless) sequenced from 83 exemplar taxa representing 18 planthopper families and outgroups. Data sets were analyzed separately and in various combinations under the maximum parsimony criterion, and the total combined dataset was analyzed via both maximum parsimony and partitioned Bayesian criteria; results of the combined analyses were concordant across reconstruction paradigms. Relationships recovered suggest several major planthopper lineages, including: (1) Delphacidae+Cixiidae; (2) Kinnaridae+Meenoplidae; (3) Fulgoridae+Dictyopharidae; (4) Lophopidae+Eurybrachidae (possibly+Flatidae); (5) Ricaniidae+Caliscelidae (possibly+Tropiduchidae). Results also suggest the placement of Achilixiidae outside of Cixiidae and of Tettigometridae as one of the more recently diversified lineages within Fulgoroidea. The resulting phylogeny supports Asche's (1987) hypothesis of a functional trend in ovipositor structure across families.     

Endosymbiotic microorganisms in Adelges (Sacchiphantes) viridis (Insecta,Hemiptera, Adelgoidea: Adelgidae): Molecular characterization,ultrastructure and transovarial transmission

The aim of this paper was to identify endosymbiotic microorganisms living in the body cavity of a Polish population of an aphid, Adelges (Sacchiphantes) viridis, as well as to describe their ultrastructure and mode of transmission between generations. Molecular data (amplification and sequencing of 16S rRNA genes) indicated that endosymbionts of A. (S.) viridis are Betaproteobacteria of the species “Candidatus Vallotia virida”. Endosymbiotic bacteria are rod-shaped and localized in the cytoplasm of specific cells, termed bacteriocytes, of host insects. Endosymbionts sharing the same bacteriocytes differ in the density of their cytoplasm. There are two morphotypes of endosymbiotic bacteria: with electron-dense cytoplasm and electron-translucent cytoplasm. Since only bacteria containing electron-dense cytoplasm were observed in the binary fusion stage, differences in density of the cytoplasm are probably due to changes in the cytoskeleton of bacteria during division. Endosymbionts of A. (S.) viridis are transovarially (i.e. via oocytes) transmitted from the mother to the offspring.     

Predators associated with the hemlock woolly adelgid (Hemiptera: Adelgidae) in the Pacific Northwest

The hemlock woolly adelgid, Adelges tsugae Annand (Hemiptera: Adelgidae), is causing widespread mortality of eastern hemlock, Tsuga canadensis L. Carrière, in the eastern United States. In western North America, feeding by A. tsugae results in negligible damage to western hemlock, Tsuga heterophylla (Raf.) Sargent. Host tolerance and presence of endemic predators may be contributing to the relatively low levels of injury to T. heterophylla caused by A. tsugae. Field surveys of the predator community associated with A. tsugae infestations on 116 T. heterophylla at 16 sites in Oregon and Washington were conducted every 4-6 wk from March 2005 through November 2006. Fourteen uninfested T. heterophylla were also surveyed across 5 of the 16 sites. Each sample tree was assigned an A. tsugae population score ranging from 0 to 3. Predators collected from A. tsugae-infested T. heterophylla represent 55 species in 14 families, listed in order of abundance: Derodontidae, Chamaemyiidae, Hemerobiidae, Coccinellidae, Cantharidae, Reduviidae, Miridae, Syrphidae, Chrysopidae, Coniopterygidae, Staphylinidae, Anthocoridae, Nabidae, and Raphidiidae. Laricobius nigrinus Fender (Coleoptera: Derodontidae), Leucopis argenticollis Zetterstedt (Diptera: Chamaemyiidae), and Leucopis atrifacies (Aldrich) (Chamaemyiidae) were the most abundant predators; together comprising 59% of predator specimens recovered. Relationships among predators and A. tsugae were determined through community structure analysis. The abundances of Laricobius spp. larvae, L. nigrinus adults, Leucopis spp. larvae, and L. argenticollis adults were found to be positively correlated to A. tsugae population score. Predators were most abundant when the two generations of A. tsugae eggs were present. L. argenticollis and L. atrifacies were reared on A. tsugae in the laboratory, and host records show them to feed exclusively on Adelgidae.     

蜡蝉总科(昆虫纲:半翅目)系统分类研究进展

蜡蝉总科隶属于半翅目头喙亚目,大致可分为18～21个科。中国记载16科。蜡蝉总科的科级系统发育关系一直存在着广泛争议,至今尚无一个为大多数学者所接受的分类系统。因此,研究蜡蝉总科的系统发育具有重要的理论价值。本文从种及种下阶元的分类鉴定、种上阶元的系统发育、生物地理学等方面对蜡蝉总科的分类研究进行综述。     

Bacteriocyte-associated gammaproteobacterial symbionts of the Adelges nordmannianae/piceae complex (Hemiptera: Adelgidae)

Adelgids (Insecta: Hemiptera: Adelgidae) are known as severe pests of various conifers in North America, Canada, Europe and Asia. Here, we present the first molecular identification of bacteriocyte-associated symbionts in these plant sap-sucking insects. Three geographically distant populations of members of the Adelges nordmannianae/piceae complex, identified based on coI and ef1alpha gene sequences, were investigated. Electron and light microscopy revealed two morphologically different endosymbionts, coccoid or polymorphic, which are located in distinct bacteriocytes. Phylogenetic analyses of their 16S and 23S rRNA gene sequences assigned both symbionts to novel lineages within the Gammaproteobacteria sharing <92% 16S rRNA sequence similarity with each other and showing no close relationship with known symbionts of insects. Their identity and intracellular location were confirmed by fluorescence in situ hybridization, and the names ‘Candidatus Steffania adelgidicola'' and ‘Candidatus Ecksteinia adelgidicola'' are proposed for tentative classification. Both symbionts were present in all individuals of all investigated populations and in different adelgid life stages including eggs, suggesting vertical transmission from mother to offspring. An 85 kb genome fragment of ‘Candidatus S. adelgidicola'' was reconstructed based on a metagenomic library created from purified symbionts. Genomic features including the frequency of pseudogenes, the average length of intergenic regions and the presence of several genes which are absent in other long-term obligate symbionts, suggested that ‘Candidatus S. adelgidicola'' is an evolutionarily young bacteriocyte-associated symbiont, which has been acquired after diversification of adelgids from their aphid sister group.     

A molecular phylogeny of fleas (Insecta: Siphonaptera): origins and host associations

Siphonaptera (fleas) is a highly specialized order of holometabolous insects comprising ～2500 species placed in 16 families. Despite a long history of extensive work on flea classification and biology, phylogenetic relationships among fleas are virtually unknown. We present the first formal analysis of flea relationships based on a molecular matrix of four loci (18S ribosomal DNA, 28S ribosomal DNA, Cytochrome Oxidase II, and Elongation Factor 1‐alpha) for 128 flea taxa from around the world representing 16 families, 25 subfamilies, 26 tribes, and 83 flea genera with eight outgroups. Trees were reconstructed using direct optimization and maximum likelihood techniques. Our analysis supports Tungidae as the most basal flea lineage, sister group to the remainder of the extant fleas. Pygiopsyllomorpha is monophyletic, as are the constituent families Lycopsyllidae, Pygiopsyllidae, and Stivaliidae, with a sister group relationship between the latter two families. Macropsyllidae is resolved as sister group to Coptopsyllidae with moderate nodal support. Stephanociricidae is monophyletic, as are the two constituent subfamilies Stephanocircinae and Craneopsyllinae. Vermipsyllidae is placed as sister group to Jordanopsylla. Rhopalopsyllidae is monophyletic as are the two constituent subfamilies Rhopalopsyllinae and Parapsyllinae. Hystrichopsyllidae is paraphyletic with Hystrichopsyllini placed as sister to some species of Anomiopsyllini and Ctenopariini placed as sister to Carterettini. Ctenophthalmidae is grossly paraphyletic with the family broken into seven lineages dispersed on the tree. Most notably, Anomiopsyllini is paraphyletic. Pulicidae and Chimaeropsyllidae are both monophyletic and these families are sister groups. Ceratophyllomorpha is monophyletic and includes Ischnopsyllidae, Ceratophyllidae, and Leptopsyllidae. Leptopsyllidae is paraphyletic as are its constituent subfamilies Amphipsyllinae and Leptopsyllinae and the tribes Amphipsyllini and Leptopsyllini. Ischnopsyllidae is monophyletic. Ceratophyllidae is monophyletic, with a monophyletic Dactypsyllinae nested within Ceratophyllinae, rendering the latter group paraphyletic. Mapping of general host associations on our topology reveals an early association with mammals with four independent shifts to birds. © The Willi Hennig Society 2008.     

Distribution and abundance of Adelges tsugae (Hemiptera: Adelgidae) within hemlock trees

We studied the distribution of hemlock woolly adelgid, Adelges tsugae Annand (Hemiptera: Adelgidae), within hemlock trees for three summer (progrediens) and two winter (sistens) generations in northern Georgia. Eastern hemlock, Tsuga canadensis (L.) Carrière, trees were treated with 0, 10, or 25% of 1.5 g of imidacloprid per 2.5 cm of tree diameter at breast height and fertilized or not in a factorial design. Adelgid ovisacs per centimeter of branch were more abundant from June 2007 to June 2008 in the upper tree crown of insecticide untreated trees and when all trees were combined and that was the general trend for most comparisons. However, ovisacs were more abundant in the lower crown of insecticide treated trees in June 2008. More sistens nymphs settled on the upper crown branches than on the lower branches in summers 2007 and 2008. Higher eggs per ovisac were observed in the upper crown in February 2008 and in both the winter and summer 2009. In contrast, adelgids were more fecund in the lower crown in June 2008. On fertilized trees, eggs laid per adult were higher in the upper crown in February 2008. In summer 2008, eggs per ovisac were higher in the lower crown, but this reversed again to the upper crown by summer 2009. New growth of branches also varied among sample dates. These data demonstrate the variable distribution of adelgid and hemlock growth within trees over time and suggest that sampling only one crown area will not provide accurate estimates of adelgid densities.     

Phylogeny of the New World Dysdercus (Insecta: Hemiptera: Pyrrhocoridae) and Evolution of their Colour Patterns

Evolution of colour patterns is reconstructed in the case of the New World species of Dysdercus (Hemiptera: Pyrrhocoridae). The phylogenetic species concept was applied to this group, elevating five subspecific taxa to specific rank (D. capitatus, D. discolor, D. fervidus, D. luteus, and D. neglectus), and the resulting 41 phylogenetic species were analysed cladistically. Using optimization methods, evolution of colour patterns of the group is hypothesized, and possible developmental mechanisms of colour-pattern evolutionary changes are inferred.     

B chromosomes in Sternorrhyncha (Hemiptera, Insecta)

In the hemipteroid insects of the suborder Sternorrhyncha, B chromosomes are relatively common in comparison with other suborders of Hemiptera. However, the occurrence of supernumerary chromosomes is restricted, in most cases, to several genera or closely related species. At least in some species of Psylloidea with the XY sex determination system, a mitotically stable B chromosome integrated into an achiasmatic segregation system with the X, and became fixed as a Y chromosome. In some Aphidoidea with a multiple X system of sex determination, B chromosomes appear to be in fact non-functional X chromosomes. Supernumerary chromosomes thus probably play an important role in the evolution of sex determination systems in Sternorrhyncha.     

Phylogeny of the treehoppers (Insecta: Hemiptera: Membracidae): evidence from two nuclear genes

We present a molecular systematic investigation of relationships among family-group taxa of Membracidae, comprising nearly 3.5 kb of nucleotide sequence data from the nuclear genes elongation factor-1alpha (EF-1alpha: 958 bp) and 28S ribosomal DNA (28S rDNA: 2363 bp); data partitions are analyzed separately and in combination for 79 taxa. Analysis of the combined sequence data provided a better-resolved and more robust hypothesis of membracid phylogeny than did separate analyses of the individual genes. Results support the monophyly of the family Membracidae and indicate the presence of two major lineages (Centrotinae + Stegaspidinae + Centrodontinae and Darninae + Membracinae + Smiliinae). Within Membracidae, molecular data support the following assertions: (1) the previously unplaced genera Antillotolania and Deiroderes form a monophyletic group with Microcentrini; (2) Centrodontini and Nessorhinini are monophyletic clades that arise independently from within the Centrotinae; (3) Centrotinae is paraphyletic with respect to Centrodontinae; (4) the subfamily Membracinae is monophyletic and possibly allied with the darnine tribe Cymbomorphini; (5) the subfamily Darninae is paraphyletic; (6) the subfamily Smiliinae is paraphyletic, with molecular evidence indicating the exclusion of Micrutalini and perhaps Acutalini and Ceresini; and (7) Membracidae arose and diversified in the New World with multiple subsequent colonizations of the Old World. Our phylogenetic results suggest that morphology-based classifications of the Membracidae need to be reevaluated in light of emerging molecular evidence.     

DNA barcoding of genus Toxoptera Koch (Hemiptera: Aphididae): Identification and molecular phylogeny inferred from mitochondrial COI sequences

Abstract Identification of aphid species is always difficult due to the shortage of easily distinguishable morphological characters. Aphid genus Toxoptera consists of species with similar morphology and similar to Aphis in most morphological characters except the stridulatory apparatus. DNA barcodes with 1 145 bp sequences of partial mitochondrial cytochrome‐coxidase I (COI) genes were used for accurate identification of Toxoptera. Results indicated mean intraspecific sequence divergences were 1.33%, whereas mean interspecific divergences were greater at 8.29% (0.13% and 7.79% if T. aurantii 3 and T. aurantii 4 are cryptic species). Sixteen samples were distinguished to four species correctly by COI barcodes, which implied that DNA barcoding was successful in discrimination of aphid species with similar morphology. Phylogenetic relationships among species of this genus were tested based on this portion of COI sequences. Four species of Toxoptera assembled a clade with low support in maximum‐parsimony (MP) analysis, maximum‐likelihood (ML) analysis and Bayesian phylogenetic trees, the genus Toxoptera was not monophyletic, and there were two sister groups, such as T. citricidus and T. victoriae, and two clades of T. aurantii which probably presented cryptic species in the genus.     

Biogeography of the wax scales (Insecta: Hemiptera: Coccidae: Ceroplastinae)

The modern and native distributions of wax scales are documented and an area cladogram including seventy species is presented. Wax scales are distributed worldwide, but most species are native to either South America or Africa. Their native distribution pattern is discussed in relation to their host-plant specificity and to the dispersal and vicariance theories of biogeography. The vicariance theory is preferred, because the pattern can be explained satisfactorily by plate tectonics but not by dispersal from a centre of origin. The wax scale group probably originated in the combined South American–African continent at least 97 million yr ago.     

Phylogeny of the lice (Insecta, Phthiraptera) inferred from small subunit rRNA

There has been much argument about the phylogenetic relationships of the four suborders of lice (Insecta: Phthiraptera). Lyal's study of the morphology of lice indicated that chewing/biting lice (Mallophaga) are paraphyletic with respect to sucking lice (Anoplura). To test this hypothesis we inferred the phylogeny of 33 species of lice from small subunit (SSU) rRNA sequences (18S rRNA). Liposcelis sp. from the Liposcelididae (Psocoptera) was used for outgroup reference. Phylogenetic relationships among the four suborders of lice inferred from these sequences were the same as those inferred from morphology. The Amblycera is apparently the sister-group to all other lice whereas the Rhynchophthirina is apparently sister to the Anoplura; these two suborders are sister to the Ischnocera, i.e. (Amblycera (Ischnocera (Anoplura, Rhynchophthirina))). Thus, the Mallophaga (Amblycera, Ischnocera, Rhynchophthirina) is apparently paraphyletic with respect to the Anoplura. Our analyses also provide evidence that: (i) each of the three suborders of lice that are well represented in our study (the Amblycera, Ischnocera, and Anoplura) are monophyletic; (ii) the Boopiidae is monophyletic; (iii) the genera Heterodoxus and Latumcephalum (Boopiidae) are more closely related to one another than either is to the genus Boopia (also Boopiidae); (iv) the Ricinidae and Laemobothridae may be sister-taxa; (v) the Philopteridae may be paraphyletic with respect to the Trichodectidae; (vi) the genera Pediculus and Pthirus are more closely related to each other than either is to the genus Pedicinus ; and (vii) in contrast to published data for mitochondrial genes, the rates of nucleotide substitution in the SSU rRNA of lice are not higher than those of other insects, nor do substitution rates in the suborders differ substantially from one another.     

Revision of New Zealand Orthotylinae (Insecta: Hemiptera: Miridae)

Abstract

Six genera and 11 species of Orthotylinae are now known from New Zealand. Zanchius Distant is newly recorded. One new genus, Tridiplous, and seven new species of endemic New Zealand Orthotylini are described and illustrated. Four new species are described in Tridiplous: T. burrus, T. parvapiatus, T. penmani, and T. virens, and three new species are described in Zanchius: Z. ater, Z. rubicrux, and Z. totus. Keys are provided to identify New Zealand taxa of the tribes of Orthotylinae, genera and species of Halticini, genera of Orthotylini, and species of Tridiplous and Zanchius. The bugs are illustrated with colour habitus photos, and drawings including male and female genitalia. Economic importance is discussed.     

Tettigarctidae (Insecta: Hemiptera: Cicadoidea) from the Middle Jurassic of Inner Mongolia, China

Three new species of Tettigarctidae from the Middle Jurassic of Daohugou, China are described: Shuraboprosbole daohugouensis nov. sp., S. minuta nov. sp., and S. media nov. sp. The revised diagnostic characters of Shuraboprosbole Becker-Migdisova are provided based on well-preserved whole-bodied fossil tettigarctids. So far, only four definitive species of Tettigarctidae are recorded from the Mesozoic of China. The pronotum concealing major of mesonotum seems to be an autapomorphy of Tettigarctinae. The “cicadellid-like tarsi” found in Cicadellidae and Brazilian Tettigarctidae are probably a parallel evolution.     

Effects of fertilizer and low rates of imidacloprid on Adelges tsugae (Hemiptera: Adelgidae)

Healthy hemlock trees, Tsuga canadensis (L.) Carrière, and hemlock woolly adelgid, Adelges tsugae Annand (Hemiptera: Adelgidae), populations should favor retention and population growth of adelgid predators such as Laricobius nigrinus Fender (Coleoptera: Derodontidae) and Sasajiscymnus tsugae (Sasaji & McClure) (Coleoptera: Coccinellidae). Eastern hemlock trees between 15 and 38 cm diameter at breast height (dbh) were treated with 0, 10, or 25% of 1.5 g imidacloprid (Merit 75 WP) per 2.5 cm dbh and were either fertilized or not, in a 3 by 2 factorial design. After 2 yr, imidacloprid reduced the numbers of ovisacs and eggs found on trees in a dosage-dependent manner, while enhancing tree growth parameters such as new shoots or needles and the length of new shoots. Fertilized trees had greater adelgid fecundity, which was positively correlated with total foliar N in both winter generations. In February 2009 (27 mo after imidacloprid treatment), higher imidacloprid dosages to unfertilized trees resulted in reduced adelgid fecundity. Concentrations of N, P, and K were higher in the foliage of trees treated with insecticide, whereas foliar aluminum concentrations were consistently lower in trees with higher insecticide dosages. Trees treated with low rates of imidacloprid were healthier than untreated trees, but only trees treated with the 0.1 x dosage had sufficient adelgids to possibly sustain predators over extended periods.     

Speciation by host-switching in pinyon Cinara (Insecta: Hemiptera: Aphididae)

Parasite-host cospeciation has received much attention as an important mechanism in the diversification of phytophagous insects. However, studies have shown that for certain taxa, it is not host fidelity but host-switching that plays the critical role in speciation. Cinara are aphids (Insecta: Hemiptera: Aphididae: Lachninae) that feed exclusively on the woody parts of conifers of the Cupressaceae and Pinaceae. They are unusual aphids because most Pinaceae play host to several species of Cinara. The aphids show relatively strong host fidelity, and as a consequence historically have been treated based on the taxonomy of their hosts. The historical paradigm of aphid evolution implies that Cinara species have radiated to different parts of the same host species and/or speciated with their host. Using mitochondrial cytochrome oxidase 1 and nuclear elongation factor 1-alpha DNA sequences, we performed molecular phylogenetic analysis of Cinara species, concentrating on those associated with pinyon pines in the southwestern USA. We determined that switching hosts has played a key role in the speciation of the genus, reflected in the polyphyly of pinyon-feeding Cinara. Furthermore, species sharing a common feeding site on different hosts were more closely related to each other than to those sharing the same host but at different feeding sites, suggesting that feeding site fidelity plays a more important role in speciation than does host fidelity in general. This study also elucidated the primary taxonomy of various species: it suggested that Cinara rustica Hottes is a junior synonym of C. edulis (Wilson) and that C. wahtolca Hottes represents two species on the two different pinyon pine species, Pinus edulis Englem. and P. monophylla Torr. & Frem.     

Evolution of specialization: a phylogenetic study of host range in the red milkweed beetle (Tetraopes tetraophthalmus)

Specialization is common in most lineages of insect herbivores, one of the most diverse groups of organisms on earth. To address how and why specialization is maintained over evolutionary time, we hypothesized that plant defense and other ecological attributes of potential host plants would predict the performance of a specialist root-feeding herbivore (the red milkweed beetle, Tetraopes tetraophthalmus). Using a comparative phylogenetic and functional trait approach, we assessed the determinants of insect host range across 18 species of Asclepias. Larval survivorship decreased with increasing phylogenetic distance from the true host, Asclepias syriaca, suggesting that adaptation to plant traits drives specialization. Among several root traits measured, only cardenolides (toxic defense chemicals) correlated with larval survival, and cardenolides also explained the phylogenetic distance effect in phylogenetically controlled multiple regression analyses. Additionally, milkweed species having a known association with other Tetraopes beetles were better hosts than species lacking Tetraopes herbivores, and milkweeds with specific leaf area values (a trait related to leaf function and habitat affiliation) similar to those of A. syriaca were better hosts than species having divergent values. We thus conclude that phylogenetic distance is an integrated measure of phenotypic and ecological attributes of Asclepias species, especially defensive cardenolides, which can be used to explain specialization and constraints on host shifts over evolutionary time.     

Characteristics of the sperm structure in heteroptera (Hemiptera,Insecta)

Spermatozoa from eight heteropterans, each representing a different family, have been examined by electron microscopy in order to determine whether there exist characters typical for this insect group. Two such characters were found, namely bridges from the mitochondrial derivatives to the axonemal microtubules nos. 1 and 5, and two or three, rather than one, crystalline bodies within the mitochondrial derivatives. It is suggested that these characters are synapomorphic traits. The heteropteran spermatozoa lack accessory bodies typical of spermatozoa from many related groups of insects. The acrosome of the aquatic or semi-aquatic heteropterans (the infraorders Nepomorpha and Gerromorpha) has a peculiar inner structure consisting of tightly packed tubules. On the common theme of the heteropteran sperm structure, there were many variations, and the spermatozoa of each species examined can be recognized.     

Evolution in Aeschynanthus (Gesneriaceae) inferred from ITS sequences

 Aeschynanthus Jack, an epiphytic genus with c.160 species, is widespread in SE Asia. We selected 50 species for ITS nrDNA sequencing, to include all biogeographic areas and all infrageneric groupings, which are currently based on seed morphology. Some species were sequenced directly from PCR product; others cloned because of ITS length polymorphisms. The clone sequences were analysed individually and combined in an elision matrix. Results extend earlier findings that Aeschynanthus is divided into two clades, one occurring primarily in mainland SE Asia and the other in Malesia. This pattern is interpreted as indicating an ancient vicariance event followed by dispersal and plate fusion. Clade I has straight or clockwise spiral orientation of the testa cells and clade II anticlockwise spiral orientation. In clade I some species of section Microtrichium form a basal group with other sections being polyphyletic or paraphyletic. In clade II the monophyletic section Aeschynanthus is nested within the paraphyletic basal Microtrichium. Received February 8, 2001 Accepted June 8, 2001