The mitochondrial genome of Bacillus stick insects (Phasmatodea) and the phylogeny of orthopteroid insects

The Order Phasmatodea (stick and leaf insects) includes many well-known species of cryptic phytophagous insects. In this work, we sequenced the almost complete mitochondrial genomes of two stick insect species of the genus Bacillus. Phasmatodea pertain to the Polyneoptera, and represent one of the major clades of heterometabolous insects. Orthopteroid insect lineages arose through rapid evolutionary radiation events, which likely blurred the phylogenetic reconstructions obtained so far; we therefore performed a phylogenetic analysis to resolve and date all major splits of orthopteroid phylogeny, including the relationships between Phasmatodea and other polyneopterans. We explored several molecular models, with special reference to data partitioning, to correctly detect any phylogenetic signal lying in rough data. Phylogenetic Informativeness analysis showed that the maximum resolving power on the orthopteroid mtDNA dataset is expected for the Upper Cretaceous, about 80millionyears ago (Mya), but at least 70% of the maximum informativeness is also expected for the 150-200 Mya timespan, which makes mtDNA a suitable marker to study orthopteroid splits. A complete chronological calibration has also been computed following a Penalized Likelihood method. In summary, our analysis confirmed the monophyly of Phasmatodea, Dictyoptera and Orthoptera, and retrieved Mantophasmatodea as sister group of Phasmatodea. The origin of orthopteroid insects was also estimated to be in the Middle Triassic, while the order Phasmatodea seems to appear in the Upper Jurassic. The obtained results evidenced that mtDNA is a suitable marker to unravel the ancient splits leading to the orthopteroid orders, given a proper methodological approach.     

Species delimitation in the crypsis‐defended and polymorphic stick insects of the genus Libethra (Phasmatodea,Diapheromeridae)

Crypsis is a widespread defensive mechanism for prey ambushed by visually oriented predators. One well‐documented consequence of this interaction is the formation of search images by the predator that leads to the coexistence of multiple morphs in the prey population. Libethra is a genus of stick insects whose ornamentation and coloration are decidedly protective and exhibit exuberant intraspecific variation. Despite this fact, these traits have been extensively used to support the boundaries between the species it includes. This is the case in the north Andean species Libethra rabdota and Libethra rabdotula, delimited on the basis of a specific arrangement of ornamental characters. To state whether such taxonomical practice is meaningful and convenient, we evaluated the grouping of specimens of both species by means of the independent inference of phylogenies obtained with mitochondrial DNA sequences and ootaxonomy. This article synonymizes L. rabdota and L. rabdotula, redescribes the adults and describes the egg of the species. Additionally, we demonstrate that the characters related to crypsis used for the diagnosis of L. rabdota are not exclusive to this species by describing Libethra rioblanco sp. n. and Libethra ucumariensis sp. n. Our findings suggest that this genus requires extensive revision, but also provide support for the usage of different tools that might produce more accurate and stable classification. Evolutionary scenarios for both the extreme morphological variation and possible homoplastic state of camouflage‐related traits are discussed herein.     

Limited effects of above‐ and belowground insects on community structure and function in a species‐rich grassland

Question: Do above‐ and belowground insects differentially impact plant community structure and function in a diverse native grassland? Location: Rough fescue prairie in Alberta, Canada. Methods: Above‐ and belowground insects were suppressed with insecticides for 5 years using a randomised block design. During this experiment, a severe drought began in 2001 and ended in 2003. Aboveground plant growth was measured as cover and biomass from 2001 to 2005. Root demography was measured in 2002 using a minirhizotron. Mixed models and repeated measures ANOVA were used to determine treatment effects on a number of response variables. MRBP were used to test for treatment effects on community composition. Results: Five years of insect suppression had few significant effects on plant growth, species richness or community composition, and were limited primarily to the severe drought in 2002. During the drought, insect attack increased root mortality, reduced plant cover, and altered community composition. Following the drought, plant responses were unaffected by insecticide application for the remainder of the experiment. Conclusions: Five years of insect suppression had only minor effects on community structure and function in this diverse native grassland. There was no indication that these effects increased over time. The results are counter to studies conducted in productive old‐field communities that revealed large effects of insects on community structure. We suggest that the unique features of this system, such as high species evenness, abundance of generalist herbivores, and a lack of competition for light among plants, limited the potential for insects to greatly impact community‐level processes.     

Molecular phylogeny of parabasalids based on small subunit rRNA sequences, with emphasis on the Trichomonadinae subfamily

We determined small subunit ribosomal DNA sequences from three parabasalid species, Trichomitus batrachorum strain R105, Tetratrichomonas gallinarum, and Pentatrichomonas hominis belonging to the Trichomonadinae subfamily. Unrooted molecular phylogenetic trees inferred by distance, parsimony, and likelihood methods reveal four discrete clades among the parabasalids. The Trichomonadinae form a robust monophyletic group. Within this subfamily T. gallinarum is closely related to Trichomonas species as supported by morphological data, with P. hominis and Pseudotrypanosoma giganteum occupying basal positions. Our analysis does not place T. batrachorum within the Trichomonadinae. Trichomitus batrachorum (strains R105 and BUB) and Hypotrichomonas acosta form a well-separated cluster, suggesting the genus Trichomitus is polyphyletic. The emergence of T. batrachorum precedes the Trichomonadinae-Tritrichomonadinae dichotomy, emphasizing its pivotal evolutionary position among the Trichomonadidae. A third cluster unites the Devescovinidae and the Calonymphidae. The fourth clade contains the three hypermastigid sequences from the genus Trichonympha, which exhibit the earliest emergence among the parabasalids. The addition of these three new parabasalid species did not however resolve ambiguities regarding the relative branching order of the parabasalid clades. The phylogenetic positions of Tritrichomonas faetus, Monocercomonas sp., Dientamoeba fragilis, and the unidentified Reticulitermes flavipes gut symbiont 1 remain unclear.     

Developmental instability, hybridization and heterozygosity in stick insects of the genus Bacillus (Insecta; Phasmatodea) with different modes of reproduction

Several genetic factors are assumed to influence developmental instability (DI). One is the level of heterozygosity, with higher levels often being associated with decreased DI; another is genetic incompatibility in hybrids, which in several cases has been shown to increase DI. The genus Bacillus includes species which have both amphigonic heterozygous reproducing populations and homozygous parthenogenetic reproducing populations (B. rossius rossius and B. r. redtenbacheri). Furthermore, Bacillus includes hybrid parthenogenetic species, which have very high levels of almost fixed heterozygosities (B. atticus, B. whitei, B. lynceorum). We investigated the phenotypic variance (σ²p) and the impact of hybridization and level of heterozygosity on DI in females from these populations and species of Bacillus. DI was estimated as fluctuating asymmetry (FA) for three bilateral traits: the labial palpus, the maxillary palpus and the antenna. For the labial palpus and maxillary palpus we found, in general, a lower level of DI in the amphigonic females compared with parthenogenetic counterparts from the same species and with parthenogenetic females from the three hybrid species. A higher DI of the antenna was found in the hybrid species when compared with both parthenogenetic and amphigonic populations of the nonhybrid species, suggesting that the genes controlling antenna development are located on the sex chromosomes. The development of the investigated bilateral characters in the hybrid species seemed to be affected more by factors relating to genetic incompatibilities as a consequence of hybridization than by the stabilizing force of increased heterozygosity. Only few differences in σ²p were observed, supporting the possibility that the observed differences in DI are related mainly to internal genetic factors. © 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 87 , 249–259.     

A molecular framework for the phylogeny of the ant subfamily dolichoderinae

Partial sequences are reported for the mitochondrial genes for cytochrome oxidase subunits 2 and 3 and for cytochrome b, and the entire sequence of the gene for tRNA(Leu)(UUR) for species from 14 genera of dolichoderine ants and from three outgroup genera. Considerable variation was observed between tRNA genes in the size of the TPsiC arm and the DHU and anticodon loops and whether or not the TPsiC stem possesses a GC pair. The outgroup taxa showed complete TAA CO1 stop codons, but dolichoderines have either TA or T. The outgroup taxa showed a noncoding gap between the CO1 and the tRNA(Leu)(UUR) genes. A phylogeny-independent compatibility test using the amino acid sequences showed differences between the genes consistent with variation in evolutionary rates, according with other studies. Base compositions proved heterogeneous between species, hence phylogenetic analysis was restricted to the protein sequences using maximum likelihood and the mtREV24 replacement matrix. A maximum-likelihood consensus tree has similarities to those from morphological studies with some exceptions such Leptomyrmex falling within the dolichoderine genera rather than basally, and the accretion of genera formerly included under Iridomyrmex. Features of the tRNA genes and the CO1 termination codons agree quite well with the molecular phylogeny.     

The phylogenetic placement and biogeographical origins of the New Zealand stick insects (Phasmatodea)

The Lanceocercata are a clade of stick insects (Phasmatodea) that have undergone an impressive evolutionary radiation in Australia, New Caledonia, the Mascarene Islands and areas of the Pacific. Previous research showed that this clade also contained at least two of the nine New Zealand stick insect genera. We have constructed a phylogeny of the Lanceocercata using 2277 bp of mitochondrial and nuclear DNA sequence data to determine whether all nine New Zealand genera are indeed Lanceocercata and whether the New Zealand fauna is monophyletic. DNA sequence data were obtained from mitochondrial cytochrome oxidase subunits I and II and the nuclear large subunit ribosomal RNA and histone subunit 3. These data were subjected to Bayesian phylogenetic inference under a partitioned model and maximum parsimony. The resulting trees show that all the New Zealand genera are nested within a large New Caledonian radiation. The New Zealand genera do not form a monophyletic group, with the genus Spinotectarchus Salmon forming an independent lineage from the remaining eight genera. We analysed Lanceocercata apomorphies to confirm the molecular placement of the New Zealand genera and to identify characters that confirm the polyphyly of the fauna. Molecular dating analyses under a relaxed clock coupled with a Bayesian extension to dispersal‐vicariance analysis was used to reconstruct the biogeographical history for the Lanceocercata. These analyses show that Lanceocercata and their sister group, the Stephanacridini, probably diverged from their South American relatives, the Cladomorphinae, as a result of the separation of Australia, Antarctica and South America. The radiation of the New Caledonian and New Zealand clade began 41.06 million years ago (mya, 29.05–55.40 mya), which corresponds to a period of uplift in New Caledonia. The main New Zealand lineage and Spinotectarchus split from their New Caledonian sister groups 33.72 (23.9–45.62 mya) and 29.9 mya (19.79–41.16 mya) and began to radiate during the late Oligocene and early Miocene, probably in response to a reduction in land area and subsequent uplift in the late Oligocene and early Miocene. We discuss briefly shared host plant patterns between New Zealand and New Caledonia. Because Acrophylla sensu Brock & Hasenpusch is polyphyletic, we have removed Vetilia Stål from synonymy with Acrophylla Gray.     

A new genus and new species of Philippine stick insects (Insecta: Phasmatodea) and phylogenetic considerations

Based on characters of both sexes, a new genus and species of the basal euphasmatodean lineage Aschiphasmatidae is described and figured from the Philippines. Dallaiphasma eximius gen. et sp. n. displays interesting features for the group, including: a cone-shaped vertex, which is notably raised above the pronotum; the tibial area apicalis represented by a depressed membranous posterior lateral region, and a strongly sclerotized central apical region; the euplantulae consisting of smooth-type attachment pads; the pretarsal claws pectination reduced to minute denticulations; and the well-differentiated boundary between the metanotum and the first abdominal tergum. The phylogenetic information content of the new findings is discussed. Furthermore, as a result of this study, the Aschiphasmatidae are newly recorded from Mindoro island, and now include five genera and six species of the Philippines.     

A molecular phylogeny for the South African limbless lizard taxa of the subfamily Acontinae (Sauria: Scincidae) with special emphasis on relationships within Acontias

In the present study, relationships among three genera Acontias, Acontophiops, and Typhlosaurus, that comprise the South African limbless lizard subfamily Acontinae, were assessed with partial sequences of the 16S rRNA mitochondrial DNA gene. In addition, relationships within Acontias were further investigated using sequence data from the cytochrome oxidase I gene (COI). Maximum likelihood and maximum parsimony analyses of the 16S rRNA mtDNA data revealed that within this subfamily, Typhlosaurus is basal while Acontophiops and Acontias are sister taxa. Based on the 16S rRNA mtDNA data, the relationships within Acontias placed A. meleagris orientalis as the sister taxon of A. percivali tasmani, with A. m. orientalis lineacauda morph and A. m. meleagrus being the sister taxa to this group. The small-bodied skinks A. lineatus lineatus and A. l. tristis formed a monophyletic group, with the medium-bodied species A. gracilicauda gracilicauda being their sister taxon. Analyses of the COI gene for Acontias place A. m. orientalis as the sister taxon of A. p. tasmani with both A. meleagris meleagris and A. m. orientalis lineacauda being distinct. In contrast to the 16S rRNA mtDNA data, the COI data placed A. g. gracilicauda as the sister taxon to these medium-bodied species; while the subspecies status of the small-bodied taxa A. l. lineatus and A. l. tristis is reaffirmed. Combined analysis of both gene fragments for Acontias taxa recovered the same clades as found using only COI data. Systematic affinities in Acontias are discussed. These results indicate that Acontias is more species rich than previously thought.     

Ascidian molecular phylogeny inferred from mtDNA data with emphasis on the Aplousobranchiata

We explored the usefulness of mtDNA data in assessing phylogenetic relationships within the Ascidiacea. Although ascidians are a crucial group in studies of deuterostome evolution and the origin of chordates, little molecular work has been done to ascertain the evolutionary relationships within the class, and in the studies performed to date the key group Aplousobranchiata has not been adequately represented. We present a phylogenetic analysis based on mitochondrial cytochrome c oxidase subunit I (COI) sequences of 37 ascidian species, mainly Aplousobranchiata (26 species). Our data retrieve the main groups of ascidians, although Phlebobranchiata appeared paraphyletic in some analyses. Aplousobranch ascidians consistently appeared as a derived group, suggesting that their simple branchial structure is not a pleisiomorphic feature. Relationships between the main groups of ascidians were not conclusively determined, the sister group of Aplousobranchiata was the Stolidobranchiata or the Phlebobranchiata, depending on the analysis. Therefore, our data could not confirm an Enterogona clade (Aplousobranchiata+Phlebobranchiata). All of the tree topologies confirmed previous ideas, based on morphological and biochemical characters, suggesting that Cionidae and Diazonidae are members of the clade Aplousobranchiata, with Cionidae occupying a basal position within them in our analyses. Within the Aplousobranchiata, we found some stable clades that provide new data on the evolutionary relationships within this large group of ascidians, and that may prompt a re-evaluation of some morphological characters.     

Restoration prospects of abandoned species‐rich sandy grassland in Hungary

Abstract. Secondary succession and seed bank formation was studied in a formerly grazed, abandoned, eastern Hungarian sandy steppe‐meadow (Pulsatillo‐Festucetum). The vegetation was sampled at different elevations of a sand dune which became partly invaded by the tree Robinia pseudo‐acacia ca. 10 yr ago. Pre‐abandonment vegetation records were used as historic references. Though composition of the non‐invaded grassland only changed moderately, dominance of tall grasses (Elymus hispidus, Poa angustifolia) increased significantly at the cost of annuals and low stature perennials. In the stand invaded by Robinia most grassland species were lost and replaced by nitrophytes. Vertical position influenced species abundance, but affected the composition only moderately. Fine‐scale zonation of the vegetation also changed with time. Species richness of the above‐ground vegetation and the seed density of soil samples at the lower elevation were slightly greater than at the higher sites. Seed banks of sensitive grassland specialists (e.g. Pulsatilla pratensis subsp. hungarica) disappeared during grass encroachment. Following extinction from above‐ground vegetation, restoration must rely on dispersal from adjacent areas. In contrast, several annuals and perennials, which survived this degradation stage in the above‐ground vegetation, possessed seed banks. Many of these species became extinct from the vegetation during the Robinia invasion but left viable persistent seeds. This fact is promising for restoration of the Potentillo‐Festucetum sandy pasture. Competitive weedy species and sprouting Robinia can, however, limit seedling establishment.     

An improved molecular phylogeny of the Nematoda with special emphasis on marine taxa

Phylogenetic reconstructions of relations within the phylum Nematoda are inherently difficult but have been advanced with the introduction of large-scale molecular-based techniques. However, the most recent revisions were heavily biased towards terrestrial and parasitic species and greater representation of clades containing marine species (e.g. Araeolaimida, Chromadorida, Desmodorida, Desmoscolecida, Enoplida, and Monhysterida) is needed for accurate coverage of known taxonomic diversity. We now add small subunit ribosomal DNA (SSU rDNA) sequences for 100 previously un-sequenced species of nematodes, including 46 marine taxa. SSU rDNA sequences for >200 taxa have been analysed based on Bayesian inference and LogDet-transformed distances. The resulting phylogenies provide support for (i) the re-classification of the Secernentea as the order Rhabditida that derived from a common ancestor of chromadorean orders Araeolaimida, Chromadorida, Desmodorida, Desmoscolecida, and Monhysterida and (ii) the position of Bunonema close to the Diplogasteroidea in the Rhabditina. Other, previously controversial relationships can now be resolved more clearly: (a) Alaimus, Campydora, and Trischistoma belong in the Enoplida, (b) Isolaimium is placed basally to a big clade containing the Axonolaimidae, Plectidae, and Rhabditida, (c) Xyzzors belongs in the Desmodoridae, (d) Comesomatidae and Cyartonema belongs in the Monhysterida, (e) Globodera belongs in the Hoplolaimidae and (f) Paratylenchus dianeae belongs in the Criconematoidea. However, the SSU gene did not provide significant support for the class Chromadoria or clear evidence for the relationship between the three classes, Enoplia, Dorylaimia, and Chromadoria. Furthermore, across the whole phylum, the phylogenetically informative characters of the SSU gene are not informative in a parsimony analysis, highlighting the short-comings of the parsimony method for large-scale phylogenetic modelling.     

Top‐soil translocation as a technique in the re‐creation of species‐rich meadows

Abstract. In France, most civil engineering and excavation projects are at present accompanied by compensatory measures with the aim of preserving biodiversity. In order to avoid the destruction of a habitat of high conservation interest in NE France, harbouring two legally protected plant species, an experiment of soil translocation was conducted on an area of 1 ha. The donor site was an extensively managed mesophilic meadow and the receiving site was a neighbouring arable land. The vegetation of the translocated meadow was described 8 and 17 months after soil translocation, and compared (1) with vegetation resulting from more classical restoration techniques tested on the arable land (natural regeneration and seed mixture sowing) and (2) with the soil seed bank and vegetation previously present on the donor site. Results showed that the soil translocation technique permitted the development of many meadow species, including two legally protected species, and few ruderal species despite a large area of bare ground. This technique seems effective in terms of number and abundance of meadow species compared to natural regeneration and commercial seed sowing. In the case of the two classical methods, species richness was lower and only widespread species were present. Topsoil translocation provides a good compensatory method to avoid habitat and species destruction. However, the study should be continued, with the aim of assessing the longer term development and stabilization of the vegetation of the translocated meadow.     

A molecular phylogeny of apple snails (Gastropoda, Caenogastropoda, Ampullariidae) with an emphasis on African species

Ampullariids are widespread in Africa, Asia, South- and Central America, and the Caribbean Islands. Basal phylogenetic relationships of the African genera Afropomus and Saulea have been inferred based on anatomical evidence. Until recently the Viviparidae was regarded as the sister-group of Ampullariidae, but recent molecular data infer a sister-group relationship with Campanilidae. We have used members of both families as outgroups in the present investigation on ampullariid phylogeny. We have used data from portions of five molecular loci, that is, the nuclear genes 18S rRNA, 28S rRNA and H3, and the mitochondrial genes 16S rRNA and COI. Our data most often infer a basal position of Afropomus . The West African species Saulea is inferred as the basal member of a clade including the South American Marisa and Pomacea . We hypothesize that evolutionary lineages leading to Saulea and the American genera were isolated from each other by vicariance events (Gondwanaland break-up 130–110 Mya). Our individual gene analyses inferred two major clades of the African Lanistes . However, in some analyses they were not inferred as sister-groups making Lanistes paraphyletic. The African and Asian genus Pila is most often inferred to be monophyletic (except for the generally unresolved 28S). Our analyses most often inferred a sister-group relationship between Lanistes and Pila . The very low genetic diversity of the endemic radiation of Lanistes in Lake Malawi suggests that the morphological divergence has happened much faster than the molecular divergence as is also evidenced from the cichlid radiations.     

A molecular and morphological reassessment of the phylogeny of the subfamily Ophioninae (Hymenoptera: Ichneumonidae)

The phylogeny of the subfamily Ophioninae (Hymenoptera: Ichneumonidae) is investigated using molecular markers and morphological characters. We analysed the mitochondrial DNA CO1 and the nuclear 28S D2–D3 gene fragments for 74 species of Ophioninae from 25 out of the 32 recognized genera, which collectively represent 98% of described species diversity of the subfamily. Molecular markers were analysed separately and combined, with or without the adjunction of a matrix of 62 morphological characters using Bayesian inference. Our results reveal three distinct lineages, each including one of most speciose genera: Ophion, Enicospilus and Thyreodon. The comparison of the molecular data, and combined molecular plus morphological data led to the definition of the three tribes: Ophionini stat. rev. (Ophion + Alophophion + Rhopalophion + Xylophion + Afrophion); Enicospilini stat. rev. (Enicospilus + Laticoleus + Dicamptus + Hellwigiella); and Thyreodonini tribe nov. (Thyreodon + Dictyonotus + Rhynchophion). The possible association of other genera to one or another of these lineages is discussed. Ophion is a polyphyletic assemblage and requires a further revision to define the delimitation with close genera. The enigmatic Old World genus Skiapus is strongly supported as belonging to the Ophioninae, although its placement within the subfamily is ambiguous as a result of its derived genotype and phenotype. Finally, we propose a biogeographical scenario supported by this phylogeny and based on the limited available fossil data.