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Many unisexual animal lineages supposedly arose from hybridization. However, support for their putative hybrid origins mostly comes from indirect methodologies, which are rarely confirmatory. Here we provide compelling data indicating that tetraploid unisexual Calligrapha are true genetic mosaics obtained via analysis of mitochondrial DNA (mtDNA) and allelic variation and coalescence times for three single-copy nuclear genes (CPS, HARS, and Wg) in five of six unisexual Calligrapha and a representative sample of bisexual species. Nuclear allelic diversity in unisexuals consistently segregates in the gene pools of at least two but up to three divergent bisexual species, interpreted as putative parentals of interspecific hybridization crosses. Interestingly, their mtDNA diversity derives from an additional yet undiscovered older evolutionary lineage that is possibly the same for all independently originated unisexual species. One possibly extinct species transferred its mtDNA to several evolutionary lineages in a wave of hybridization events during the Pliocene, whereby descendant species retained a polymorphic mtDNA constitution. Recent hybridizations, in the Pleistocene and always involving females with the old introgressed mtDNA, seemingly occurred in the lineages leading to unisexual species, decoupling mtDNA introgression (and inferences derived from these data, such as timing and parentage) from subsequent acquisition of the new reproductive mode. These results illuminate an unexpected complexity in possible routes to animal unisexuality, with implications for the interpretation of ancient unisexuality. If the origin of unisexuality requires a mechanism where (1) hybridization is a necessary but insufficient condition and (2) multiple bouts of hybridization involving more than two divergent lineages are required, then the origins of several classical unisexual systems may have to be reassessed. © 2014 by The University of Chicago. All rights reserved., This work was funded by the Spanish Ministry of Science and Innovation (CGL2008–00007/BOS, with participation of the European Regional Development Fund), including the Formación de Personal Investigador researcher training studentship to T.M. Pilot tests focusing on ITS2 sequences were funded by the EU Synthesys initiative (FP6 Programme, Integrated Infrastructure Initiative; GB-TAF-1840) at the Natural History Museum (London).

Rapid degradation of tropical forests urges to improve our efficiency in large-scale biodiversity assessment. DNA barcoding can assist greatly in this task, but commonly used phenetic approaches for DNA-based identifications rely on the existence of comprehensive reference databases, which are infeasible for hyperdiverse tropical ecosystems. Alternatively, phylogenetic methods are more robust to sparse taxon sampling but time-consuming, while multiple alignment of species-diagnostic, typically length-variable, markers can be problematic across divergent taxa. We advocate the combination of phylogenetic and phenetic methods for taxonomic assignment of DNA-barcode sequences against incomplete reference databases such as GenBank, and we developed a pipeline to implement this approach on large-scale plant diversity projects. The pipeline workflow includes several steps: database construction and curation, query sequence clustering, sequence retrieval, distance calculation, multiple alignment and phylogenetic inference. We describe the strategies used to establish these steps and the optimization of parameters to fit the selected psbA-trnH marker. We tested the pipeline using infertile plant samples and herbivore diet sequences from the highly threatened Nicaraguan seasonally dry forest and exploiting a valuable purpose-built resource: a partial local reference database of plant psbA-trnH. The selected methodology proved efficient and reliable for high-throughput taxonomic assignment, and our results corroborate the advantage of applying ‘strict’ tree-based criteria to avoid false positives. The pipeline tools are distributed as the scripts suite ‘BAGpipe’ (pipeline for Biodiversity Assessment using GenBank data), which can be readily adjusted to the purposes of other projects and applied to sequence-based identification for any marker or taxon., This project was mainly funded by the ‘Ecology and Conservation Biology’ programme (BIOCON 08) of the Spanish private fund, ‘Fundación BBVA’ led by J.G.-Z. Additional funding was available for fieldwork thanks to Project C/032352/10 of the Interuniversity Cooperation and Research Programme (PCI-AECID 2010) of the Spanish Ministry of Foreign Affairs and Cooperation (MAEC). A.P. was funded by the postdoctoral programme ‘Juan de la Cierva’ of the Spanish Ministry of Science and Innovation (MICINN); G.D.C. by the predoctoral programme of the AECID (Spanish MAEC); and D.C. by the Knowledge Innovation Program of the Chinese Academy of Sciences (Grant No. KSXC2-EW-B-02) and the Chinese National Science Foundation (Grants No. 30870268, 31172048, J1210002).

Relatively little is known about Coleoptera genes and genomes and how these compare in different taxa. We describe here the construction, DNA sequencing and sequence comparisons of cDNA libraries from seven beetle species. A total of 6717 bacterial colonies were screened for cDNA insert containing plasmids and 2784 size selected clones were 5 ′′′ ′ - and 3 ′′ -end sequenced to produce 1620 assembled sequences. Similarity comparisons with existing protein sequence databases revealed that 65.1% had matches ( E < 10 − − − 4 ) in other organisms, with greater numbers of matches in Drosophila melanogaster than Caenorhabditis elegans and Saccharomyces cerevisiae databases. tBlastX comparisons also revealed numerous similarity hits ( E < 10 − 20 ) in intra- and interlibrary comparisons. These results show the potential of small cDNA libraries for discovery and comparative analysis of genes useful for phylogenomic and functional studies.

Leaf beetles (Chrysomelidae; ~40,000 species) are commonly solitary animals but subsociality, maternal care of broods, is known in Cassidinae and Chrysomelinae. We report 11 novel records from Brazil and Peru, bringing the number of subsocial chrysomelids to 35 species in 10 genera. Two evolutionary models of chrysomelid subsociality have been proposed. One proposed three independent origins within Chrysomelinae, based on the potential phylogenetic positions of subsocial genera. The other hypothesised that an evolutionary arms race between chrysomelid prey and their predators, parasites, and parasitoids has led to an escalation of defences. Using our phylogenies, we propose that subsociality originated independently in Cassidinae and Chrysomelinae, and several times within each subfamily. Subsociality was preceded by particular behaviours. In Cassidinae, exophagous larvae with chemically offensive faecal weaponry preceded aggregated living, group defences (e.g. cycloalexy), and maternal guarding. In Chrysomelinae, offensive glandular compounds preceded ovi- and viviparity before subsociality. © 2014 © 2014 Taylor & Francis., This study was supported by a NSF-EPSCoR grant #66928 (USA; CSC), by the Institut de Biologia Evolutiva (CSIC-UPF, Spain; JGZ), by the Centro Universitário de Lavras (Brazil; FFC), and by Stichting Bevordering van Natuurwetenschappelijk Onderzoek (Netherlands; RW).

The Cryptocephalus marginellus (Coleoptera: Chrysomelidae) complex is composed by six species that are supposed to have originated by events of allo- or parapatric speciation. In the present study we investigated the alternative hypotheses that the bacterial communities associated with six populations of this species complex are shaped by environmental factors, or reflect the proposed pattern of speciation. The microbiota associated with the six populations, from five species of the complex, have been characterized through 16S rRNA pyrotag sequencing. Based on a 97% sequence similarity threshold, data were clustered into 381 OTUs, which were analyzed using a variety of diversity indices. The microbiota of C. acquitanus and C. marginellus (Calanques) were the most diverse (over 100 OTUs), while that from C. zoiai yielded less bacterial diversity (45 OTUs). Taxonomic assignment revealed Proteobacteria, Tenericutes and Firmicutes as the dominant components of these beetles' microbiota. The most abundant genera were Ralstonia, Sphingomonas, Rickettsia, and Pseudomonas. Different strains of Rickettsia were detected in C. eridani and C. renatae. The analysis of β-diversity revealed high OTU turnover among the populations of C. marginellus complex, with only few shared species. Hierarchical clustering taking into account relative abundances of OTUs does not match the phylogeny of the beetles, therefore we hypothesize that factors other than phylogenetic constraints play a role in shaping the insects' microbiota. Environmental factors that could potentially affect the composition of bacterial communities were tested by fitting them on the results of a multi-dimensional scaling analysis. No significant correlations were observed towards the geographic distances or the host plants, while the composition of the microbiota appeared associated with altitude. The metabolic profiles of the microbiotas associated with each population were inferred from bacterial taxonomy, and interestingly, the obtained clustering pattern was consistent with the host phylogeny.

The Timarcha goettingensis complex is a monophyletic assemblage of closely related leaf beetles (Chrysomelidae), distributed from the north half of the Iberian Peninsula to Central Europe. Oligophagy, mountainous habitat and apterism are factors which are assumed to promote speciation in these beetles. We have used cytochrome oxidase subunit II mitochondrial DNA genealogies obtained from 31 sampling localities and a nested geographical distance analysis to assess the population structure and demographic factors explaining the geographical distributions of the mtDNA haplotypes in the T. goettingensis complex. The results show that there is a significant association between genetic structuring and geography. Inferences about the historical population processes in the species complex are discussed, being in general in accordance with contiguous range expansions and past fragmentations. The use of the cohesion species concept approach suggests the existence of several systematic ranks among the different T. goettingensis populations, which is in part supported by ecological traits such as trophic selection and altitudinal distribution.

Molecular clock calibration is a crucial step for placing phylogenetic trees in the temporal framework required to test evolutionary hypotheses and estimate evolutionary rates. In general, most authors agree that the best approach is to incorporate multiple calibrations to avoid the risk of bias associated with a single dating source. However, the indiscriminate inclusion of as many calibration points as possible can lead to tree shape distortion and an overestimation of the variation in evolutionary rates among branches due to errors in the geological, paleontological or paleogeographic information used for dating. We present a test of congruence among calibration hypotheses to assist their filtering prior to molecular clock analysis, which we have called Bayes Factor Cluster Analysis (BFCA). This is a heuristic method based on the comparison of pairwise calibrations hypotheses by Bayes factors that allows identifying sets of congruent calibrations. We have tested BFCA through simulation using beast and mcmctree programs and analysed a real case of multiple calibration hypotheses to date the evolution of the genus Carabus (Coleoptera: Carabidae). The analyses of simulated data showed the predictability of change in Bayes factors when comparing alternative calibration hypotheses on a particular tree topology, and thus the suitability of BFCA in identifying unreliable calibrations, especially in cases with limited variation in evolutionary rates among branches. The exclusion of inconsistent calibrations as identified by BFCA produced significant changes in the estimation of divergence times and evolutionary rates in the genus Carabus, illustrating the importance of filtering calibrations before analyses. The method has been implemented in an open-source R package called bfca to simplify its application. © 2013 British Ecological Society., This work was supported by the Spanish Ministry of Science and Innovation (CGL2006/06706 and CGL2009-10906 to C.A. and J.S. and CGL2008-00007 to J.G.-Z., the latter also with support of the European Regional Development Fund). CA received support from an FPU predoctoral studentship (Spanish Ministry of Education).

In this study we use a combination of morphological, molecular (mtDNA cytochrome c oxidase subunit 1 gene), geographic and ecological (field observations and cpDNA sequences from ingested plant tissue) data to describe and diagnose a new southern Nearctic leaf beetle species of Calligrapha Chevrolat, 1836 (Chrysomelidae: Chrysomelinae): Calligrapha thermalis Gómez-Zurita, sp. nov., associated to Perymenium mendezii De Candolle (Asteraceae). This new species, restricted to the Sierra Madre Occidental, is closely related to the Texan endemic Calligrapha wickhami Bowditch, 1911 associated to Viguiera stenoloba Blake (Asteraceae) in the northern foothills of the Sierra Madre Oriental ecoregion. The evolutionary scenarios explaining this recent taxonomic split, dated in the Pleistocene, are laid out and discussed.

Recent phylogenetic studies of flea beetles (Alticinae) based on morphological or molecular data have focused on the relationship and possible paraphyly with respect to the closely related Galerucinae, while the supra-generic classification mainly dates back to the 19th century. Here, phylogenetic analysis was performed on DNA sequences for two mitochondrial (rrnL and cox1) and two nuclear (SSU and LSU rRNA) genes from 158 genera and 165 species that cover most suprageneric groups of flea beetles proposed in the older literature. Various alignment strategies and tree search methods were used to test the stability of major clades. Besides confirmation of the placement of several alticine lineages within Galerucinae, a preliminary framework for classification of the main alticine clades was obtained. It is proposed to recognize 18 groups of genera based on well-supported nodes. These include the Altica, Amphimela, Aphthona, Blepharida, Chabria, Chaetocnema, Dibolia, Disonycha, Griva, Lactica, Longitarsus, Manobia, Monoplatus, Nisotra, Oedionychis, Pentamesa, Phygasia and Pseudodera groups. These groups provide a novel perspective to the existing classification. The analysis of 14 morphological characters used in the traditional classification of Alticinae and Galerucinae revealed high levels of homoplasy with respect to the DNA-based tree, but significant hierarchical structure in most of them. Even if not unique to any particular group of genera, these traits largely corroborate the groupings established with DNA sequences. © 2011 ., This project was partially supported by the National Basic Research Program of China (973 Program) (No. 2011CB302102), the National Natural Science Foundation of China (No. 30970393, 31010103913, 31172143), the Knowledge Innovation Program of Chinese Academy of Sciences (Nos. KSCX2-YW-Z-015, KSCX2-EW-G-4 and KSCX2-EW-Z-8), and The Leverhulme Trust (F/00696/P). Deyan Ge was sponsored by the Chinese state scholarship council.

9 páginas.-- Ge, Deyan et al., Complex morphological or functional traits are frequently considered evolutionarily unique and hence useful for taxonomic classification. Flea beetles (Alticinae) are characterized by an extraordinary jumping apparatus in the usually greatly expanded femur of their hind legs that separates them from the related Galerucinae. Here, we examine the evolution of this trait using phylogenetic analysis and a time-calibrated tree from mitochondrial (rrnL and cox1) and nuclear (small subunits and large subunits) genes, as well as morphometrics of femora using elliptic Fourier analysis. The phylogeny strongly supports multiple independent origins of the metafemoral spring and therefore rejects the monophyly of Alticinae, as defined by this trait. Geometric outline analysis of femora shows the great plasticity of this structure and its correlation with the type and diversity of the metafemoral springs. The recognition of convergence in jumping apparatus now resolves the long-standing difficulties of Galerucinae–Alticinae classification, and cautions against the value of trait complexity as a measure of taxonomic significance. The lineage also shows accelerated species diversification rates relative to other leaf beetles, which may be promoted by the same ecological factors that also favour the repeated evolution of jumping as an anti-predation mechanism.

Special Issue: Insect Evolution Below the Species Level., 1. The diversity of insects in tropical forests remains poorly known, in particular regarding the critical feeding associations of herbivores, which are thought to drive species richness in these ecosystems. 2. Host records remain elusive and traditionally require labour-intensive feeding trials. A recent approach analyses plant DNA ingested by herbivorous insects; direct PCR amplification from DNA extracts from weevils (Curculionoidea) using chloroplast (trnL intron) primers was successful in 41 of 115 cases, resulting in 40 different sequences. 3. The resulting trnL intron sequences were identified against public databases to various hierarchical levels based on their position in phylogenetic trees and shown to be members of 26 plant families from different major groups of angiosperms. 4. Among the trnL intron sequences, seven pairs or triplets of close relatives (0-2 bp difference) were found which may represent intraspecific variation in the respective host plants. 5. Molecular clock calibrations of mitochondrial cox1 sequences of weevils established great distances of lineages obtained (all splits estimated >20 Mya). Distant taxa were found to feed on the same or similar hosts in some cases, showing low evolutionary conservation of host associations among deeper levels. 6. The technique provides a new means of studying species diversity and plant-herbivore interactions in tropical forests, and removes the constraints of the need for actual observations of feeding in ecological and evolutionary studies. © 2010 The Royal Entomological Society., This work has been supported by the studentships from SENACYT-Panama, CONACYT-Mexico (196749) and the European Union-Alban (E05D058103PA) to S. P. N. J. J. R. received support from the SYNTHESYS grant (GB-TAF-4394) of the European Community Research Infrastructure and J.G-Z. from the Spanish “Fundación BBVA” (project >IN2-BST-Nicaragua).

11 páginas, 3 figuras, 2 tablas., Short-sequence fragments (‘DNA barcodes’) used widely for plant identification and inventorying remain to be applied to complex biological problems. Host–herbivore interactions are fundamental to coevolutionary relationships of a large proportion of species on the Earth, but their study is frequently hampered by limited or unreliable host records. Here we demonstrate that DNA barcodes can greatly improve this situation as they (i) provide a secure identification of host plant species and (ii) establish the authenticity of the trophic association. Host plants of leaf beetles (subfamily Chrysomelinae) from Australia were identified using the chloroplast trnL(UAA) intron as barcode amplified from beetle DNA extracts. Sequence similarity and phylogenetic analyses provided precise identifications of each host species at tribal, generic and specific levels, depending on the available database coverage in various plant lineages. The 76 species of Chrysomelinae included—more than 10 per cent of the known Australian fauna—feed on 13 plant families, with preference for Australian radiations of Myrtaceae (eucalypts) and Fabaceae (acacias). Phylogenetic analysis of beetles shows general conservation of host association but with rare host shifts between distant plant lineages, including a few cases where barcodes supported two phylogenetically distant host plants. The study demonstrates that plant barcoding is already feasible with the current publicly available data. By sequencing plant barcodes directly from DNA extractions made from herbivorous beetles, strong physical evidence for the host association is provided. Thus, molecular identification using short DNA fragments brings together the detection of species and the analysis of their interactions., This work has been supported by the Spanish Ministry of Education with an FPI doctorate studentship and short-stay programme to J.A.J.R., as well as projects REN2003-03667 and CGL2006-08810 to E.P., the CSIC Intramural Project 200730I014 to J.G.-Z. and Australian Biological Resources Study funding to C.A.M.R.

6 pages, 2 figures, 2 tables.-- PMID: 18434549 [PubMed].-- PMCID: PMC2359786.-- Available online Apr 23, 2008., Water reservoirs formed by the leaf axils of bromeliads are a highly derived system for nutrient and water capture that also house a diverse fauna of invertebrate specialists. Here we investigate the origin and specificity of bromeliad-associated insects using Copelatinae diving beetles (Dytiscidae). This group is widely distributed in small water bodies throughout tropical forests, but a subset of species encountered in bromeliad tanks is strictly specialized to this habitat. An extensive molecular phylogenetic analysis of Neotropical Copelatinae places these bromeliadicolous species in at least three clades nested within other Copelatus. One lineage is morphologically distinct, and its origin was estimated to reach back to 12–23 million years ago, comparable to the age of the tank habitat itself. Species of this clade in the Atlantic rainforest of southern Brazil and mountain ranges of northern Venezuela and Trinidad show marked phylogeographical structure with up to 8% mtDNA divergence, possibly indicating allopatric speciation. The other two invasions of bromeliad water tanks are more recent, and haplotype distributions within species are best explained by recent expansion into newly formed habitat. Hence, bromeliad tanks create a second stratum of aquatic freshwater habitat independent of that on the ground but affected by parallel processes of species and population diversification at various temporal scales, possibly reflecting the paleoclimatic history of neotropical forests., Funding was provided by German Science Foundation Grant DFG BA 2152/3-2, SYNTHESYS Grant GB-TAF 2811, the Natural History Museum London travel fund, British Ecological Society Grant SEPG 2257(a), Spanish Science Ministry Grant CGL2004-00028, and the Alexander von Humboldt Foundation.

The leaf beetle genus Cyrtonus Latreille, 1829 (Chrysomelidae, Chrysomelinae) is a species-rich genus subendemic to the Iberian Peninsula, with only a few species in the neighbouring France and Morocco. All the species are wingless and preferentially inhabiting mountainous areas. The taxonomic knowledge of this group is extremely poor and its systematics almost inexistent. Here, we analyse and characterize with a morphometric analysis one promising systematic trait, the adult body profile, distinguishing between elongated and rounded shapes. Although the monophyly of the genus is not contentious, we test for it using mitochondrial rrnl sequences and the chrysomeline homologous sequences available in GenBank. In addition, four genetic markers, two mitochondrial and two nuclear are used to produce a phylogenetic hypothesis for half the species within the genus and to analyse the evolution of shape, summarized as two continuous variables, length and width, and their ratio. These traits covary significantly with the phylogeny, showing a strong phylogenetic association: elongated species appear to constitute a clade within a paraphyletic assemblage of rounded species. In addition, the mitochondrial DNA tree is used to test for constant rate of evolution in this marker and is calibrated using both biogeographical evidence and the standard insect mitochondrial average substitution rate. This molecular clock hypothesis is used to date the age of speciation events on the phylogeny, reconstructing the origin of the genus in the Middle Miocene, with a relatively constant speciation rate until the end of the Pliocene and an apparent increase in this rate in the Pleistocene, possibly associated with the effect of dramatic climatic changes in this period. Finally, the high systematic value of shape profile in Cyrtonus is discussed, arguing the absence of evidence relating it to adaptation. © 2007 The Authors., JGZ has been funded by the project SPA/1121941 STP (Humboldt Research Fellowship, Germany), IG and EP have been supported by the projects BOS2000-0822 and REN2003-03667, Ministry of Science and Technology, Spain

Cataloging the very large number of undescribed species of insects could be greatly accelerated by automated DNA based approaches, but procedures for large-scale species discovery from sequence data are currently lacking. Here, we use mitochondrial DNA variation to delimit species in a poorly known beetle radiation in the genus Rivacindela from arid Australia. Among 468 individuals sampled from 65 sites and multiple morphologically distinguishable types, sequence variation in three mtDNA genes (cytochrome oxidase subunit 1, cytochrome b, 16S ribosomal RNA) was strongly partitioned between 46 or 47 putative species identified with quantitative methods of species recognition based on fixed unique ("diagnostic") characters. The boundaries between groups were also recognizable from a striking increase in branching rate in clock-constrained calibrated trees. Models of stochastic lineage growth (Yule models) were combined with coalescence theory to develop a new likelihood method that determines the point of transition from species-level (speciation and extinction) to population-level (coalescence) evolutionary processes. Fitting the location of the switches from speciation to coalescent nodes on the ultrametric tree of Rivacindela produced a transition in branching rate occurring at 0.43 Mya, leading to an estimate of 48 putative species (confidence interval for the threshold ranging from 47 to 51 clusters within 2 logL units). Entities delimited in this way exhibited biological properties of traditionally defined species, showing coherence of geographic ranges, broad congruence with morphologically recognized species, and levels of sequence divergence typical for closely related species of insects. The finding of discontinuous evolutionary groupings that are readily apparent in patterns of sequence variation permits largely automated species delineation from DNA surveys of local communities as a scaffold for taxonomy in this poorly known insect group.

Interspecific hybridization is a well-established cause of unisexual origins in vertebrates. This mechanism is also suspected in other apomictic taxa, but compelling evidence is rare. Here, we evaluate this mechanism and other hypotheses for the evolutionary origins of unisexuality through an investigation of Calligrapha leaf beetles. This group provides an intriguing subject for studies of unisexual evolution because it presents a rare insect example of multiple apomictic thelytokous species within a primarily bisexual genus. To investigate unisexual evolution, this study conducts the first molecular systematic analysis of Calligrapha. This involved the collection and analysis of about 3000 bp of DNA sequences--representing RNA and protein-coding loci from mitochondrial and nuclear genomes--from 54 specimens of 25 Calligrapha species, including four unisexual tetraploid taxa. Phylogenetic and molecular clock analyses indicated independent and single evolutionary origins of each of these unisexual species during the Pleistocene. Significant phylogenetic incongruence was detected between mitochondrial and nuclear datasets and found to be especially associated with the asexual taxa. This pattern is expected when unisexual lineages arise via interspecific hybridization and thus represent genetic mosaics that possess certain nuclear alleles from the paternal species lineage and mitochondrial DNA (mtDNA) alleles from the maternal parent. Analyzing the mtDNA and nuclear relatedness of unisexuals with corresponding haplotypes of bisexual Calligrapha species allowed the putative identification of these maternal and paternal species lineages for each unisexual species. Strong phenotypic similarities between unisexual taxa and their paternal parent species supported a model that involves both backcrosses of interspecific hybrids with a paternal parent and unreduced gametes. This model accounts for the origins of apomixis, polyploidy, and an overrepresentation of paternal nuclear alleles (and associated phenotypes) in unisexuals. This model is also consistent with the tetraploid karyotypes of unisexual Calligrapha, in which three sets of chromosomes (of presumed paternal ancestry) are quite morphologically homogeneous compared to the fourth. Especially intriguing was a consistent association of unisexual species with the host plant of the paternal parent but never with the maternal host. The statistical implausibility of these patterns occurring by chance further supports our inference of parental species. Moreover, it points to a potentially critical role for host-association in the formation and preservation of unisexual lineages. These findings suggest that ecological factors are critical for the diversification of unisexual as well as bisexual taxa and thus point out new research directions in the area of ecological speciation.

Previous studies of leaf beetles (Chrysomelidae) in the Timarcha goettingensis species complex using mitochondrial (cox2) and nuclear (ITS-2 rRNA) markers revealed two main clades confined to the Iberian Peninsula and the rest of Europe but showing incongruent distributions indicative of gene exchange between both groups. Because of the anastomosing nature of hybridization, which disrupts the cladistic structure of character variation, phylogenetic trees might be inappropriate to represent and study this process. Here we test for evidence of hybridization in the T. goettingensis complex by analyzing the extra homoplasy arising in hybrid genomes from the simultaneous analysis of genetically independent markers. Haplotype networks obtained by Templeton's statistical parsimony analysis were generated for combined (concatenated) cox2 and ITS-2 sequences from 167 individuals of the T. goettingensis complex. Networks were used to detect runs of homoplasious characters physically clustered along a nucleotide sequence, as evidence for recombination between both gene partitions. A hypergeometric tail probability for the chance occurrence of physically clustered character changes on the connections linking networks of genotypes was applied. The test recognized two instances of statistically significant clustering, indicating the presence of cox2-ITS-2 mosaic genotypes and reticulation of both main T. goettingensis clades, supporting the reticulate origin of samples of T. maritima in southwestern France and T. sinuatocollis/T. monserratensis in the eastern Pyrenees. Although the assessment of reticulation in DNA sequences does not provide direct proof for hybridization, the geographical distribution of mosaic genotypes in the vicinity of "pure" genotypes supports the effect of gene flow between the two divergent lineages. The study demonstrates the utility of statistical parsimony networks for the detection of hybrids in the growing number of phylogeographic studies based on multiple gene markers.

The 3400 species of Eumolpinae constitute one of the largest subfamilies of leaf beetles (Chrysomelidae). Their systematics is still largely based on late 19th century monographs and remains highly unsatisfactory. Only recently, some plesiomorphic lineages have been split out as separate subfamilies, including the southern hemisphere Spilopyrinae and the ambiguously placed Synetinae. Here we provide insight into the internal systematics of the Eumolpinae based on molecular phylogenetic analyses of three ribosomal genes, including partial mitochondrial 16S and nuclear 28S and complete nuclear 18S rRNA gene sequences. Sixteen morphological characters considered important in the higher-level systematics of Eumolpinae were also included in a combined analysis with the molecular characters. All phylogenetic analyses were performed using parsimony by optimizing length variation directly on the tree, as implemented in the POY software. The data support the monophyly of the Spilopyrinae outside the clade including all sampled Eumolpinae, corroborating their treatment as a separate subfamily within the Chrysomelidae. The systematic placement of the Synetinae remains ambiguous but consistent with considering it a different subfamily as well, since the phylogenetic analyses using all the available evidence show the representative sequence of the subfamily also unrelated to the Eumolpinae. The Megascelini, traditionally considered a separate subfamily, falls within the Eumolpinae. Several recognized taxonomic groupings within Eumolpinae, including the tribes Adoxini or Typophorini, are not confirmed by molecular data; others like Eumolpini seem well supported. Among the morphological characters analyzed, the presence of a characteristic groove on the pygidium (a synapomorphy of the Eumolpini) and the shape of tarsal claws (simple, appendiculate or bifid) stand out as potentially useful characters for taxonomic classification in the Eumolpinae.

In this work, we have analysed the karyotypes of six species of Timarcha for the first time and updated the cytological information for two additional taxa, for one of them confirming previous results (Timarcha erosa vermiculata), but not for the other (T. scabripennis). We describe the remarkable karyotype of T. aurichalcea, the lowest chromosome number in the genus (2n = 18), distinctive as well for the presence of an unusual chiasmatic sexual bivalent hitherto unreported for Timarcha. This study increases the number of species studied cytologically in this genus to forty. Additional cytogenetic analyses are performed on several species, including Ag-NOR staining and fluorescent in situ hybridization (FISH) studies with ribosomal DNA probes. Karyotype evolution is analysed by tracing different karyotype coding strategies on a published independent phylogenetic hypothesis for Timarcha based on the study of three genetic markers. The implementation of a likelihood model of character change optimized onto the phylogeny is tentatively used to detect possible drifts in chromosome changes. These analyses show that karyotype is conservative in the evolution of the genus and that there is an apparent trend to reducing chromosome number. Cytological and phylogenetic data are used to explain the evolutionary origin of the karyotype of T. aurichalcea by two centric fusions involving one pair of acrocentric autosomes and the sexual chromosomes., This work is included in the framework of projects REN2003-03667 from Spanish Ministry of Science and Technology to EP and JGZ and EC Marie Curie ERG Programme Grant (Contract no. MERG-CT-2004-500004) and project BOS2002-02870 from the Spanish Ministry of Science and Technology to JGZ

Phylogeographic analyses have mostly been based on single-gene genealogies but it is unclear how conclusions from such studies depend on the choice of gene markers. We conducted a nested geographical clade analysis [A.R. Templeton, E. Routman and C.A. Phillips (1995) Genetics 140: 767-782] based on nuclear rDNA internal transcribed spacer region 2 (ITS2) sequences in the Timarcha goettingensis species complex (Coleoptera, Chrysomelidae), and compared the inferences with an updated version of previously published results using mitochondrial cytochrome oxidase II (COIl) sequences. Inferences from ITS2 suggest that patterns of marker distribution are mostly explained by restricted gene flow with isolation by distance. In contrast, COII revealed a history of geographical structure resulting from episodic population contiguous-range expansions. Both markers also show different genealogical patterns, which are associated to the effects of genetic introgression in a putative hybrid zone between two major lineages in the complex. Altogether, these differences are attributed to distinct population and/or evolutionary dynamics of the markers, and offer a more accurate phylogeographic description for the T. goettingensis complex.

In recent years we have investigated the evolution of the Holarctic leaf-beetle genus Timarcha using molecular approaches, but to date several important questions remained unanswered, including its systematic arrangement in a temporal context, or the phylogenetic placement of the Nearctic taxa. Here I present a reanalysis of available genetic data together with newly generated data for key taxa (markers 16S rDNA, CO2, ITS-2, and 18S rDNA), including the Nearctic species (subgenus Americanotimarcha), using direct optimization-based phylogenetic reconstructions. Lineage ages are estimated using maximum likelihood branch-length estimates and the molecular clock calibration derived from several presumed vicariance events in the Mediterranean. Phylogenetic analyses and 18S rDNA divergences suggest the ancient divergence of the Nearctic and Palaearctic lineages, related to the North Atlantic opening in the middle Eocene. The diversification of the Palaearctic Timarcha seems closely related to the geological evolution of the Mediterranean area during the Tertiary, with Pleistocenic climate changes affecting species ranges and lineage extinction, but not resulting in extensive speciation.

The apterous genus Timarcha consists of three subgenera and more than 100 species in its Palearctic distribution, with specialized feeding on few plant families. Fifty-four sequences sampled from 31 taxa of the genus plus three outgroup leaf beetles were studied for their complete cytochrome oxidase II (COII) and a fragment of 16S rDNA mitochondrial genes, representing a total of about 1200 bp. Phylogenetic analyses using maximum-parsimony and distance methods for each gene separately and for the combined data set gave compatible topologies. The subgenus Metallotimarcha consistently appears in a basal position and is well differentiated from the remaining Timarcha, but no clear monophyletic grouping of Timarchostoma and Timarcha s. str. subgenera can be deduced from our analysis. Calibration of the molecular clock has been done using the opening of the Gibraltar Strait after the Messinian salinity crisis (about 5.5 MYA) as the biogeographic event causing disjunction of two particular taxa. Accordingly, the COII evolutionary rate has been estimated to be of 0.76 × 10 −8 substitution/site/year in Timarcha. Relation between phylogeny and host-plant use indicates widening of trophic regime as a derived character in Timarcha.

Internal transcribed spacer 2 (ITS2) sequences of the nuclear rDNA in forty-seven specimens (thirty-four species) of the leaf beetle genus Timarcha have been studied. Timarcha ITS2 (523 bp on average) share some sequence features with other Chrysomeloidea relatives (Chrysolina, Diabrotica and Bruchus) but have no clear similarity with any other arthropod ITS2 sequences. Interepecific divergences are in the range 0.002-0.166, and 0.124-0.206 in the comparisons between subgenera. No evidence of intragenomic divergent ITS2 sequences has been found. Secondary structures are concordant with the four-domain model proposed for vertebrates and yeast, but differs from those proposed for dipterans. Phylogenetic analysis of the ITS2 data confirms the results of a previous study based on mitochondrial sequences, as the basality of the Metallotimarcha subgenus and the absence of phylogenetic support for the Timarchostoma subgenus.

6 páginas., The first New Caledonian record for the genus Oulema Des Gozis (Coleoptera: Chrysomelidae) is reported here with the description of a new species: Oulema taophiloides sp. nov.

The study of a large number of collection specimens from the US National Museum of Natural History belonging to the genus Calligrapha Chevrolat (Chrysomelidae) from the Nearctic region has provided with one new province record and one new species record for Canada and 63 new US State records for a total of 20 taxa. These new records usually correspond to adjacent areas to already identified ranges of distribution, expanding considerably the known geographical boundaries of the species studied. The zoogeography of the genus Calligrapha in North America is discussed based on the updated information on the distribution of the genus.

The Chrysomelidae of New Caledonia are rich and unique, but insufficiently characterized. New species are being rapidly described, sometimes without careful study of the earlier taxonomic research. Karl M. Heller (Dresden, Germany) described in 1916 seven species of Eumolpinae which are redescribed here to distinguish them from other known species and to provide new information about sexual dimorphism and genitalic structures. New material is used to redefine their distribution. The males of Dematochroma lepros (Heller, 1916) and D. culminicola (Heller, 1916), and the female of D. difficilis (Heller, 1916) are described for the first time. Male and female genitalia are first described for seven and five species, respectively. D. difficilis (Heller, 1916) stat. rev., is revalidated from previous synonymy with D. terastiomerus (Heller, 1916). Lectotypes are designated for D. humboldtiana and D. terastiomerus.

Keywords. Cerambycidae, Chrysomelidae, Curculionidae, development genes, diapause genes, gene databases, genomics, insect resistance genes, phytophagy genes Abstract. Fifteen years after the publication of the first genomic sequence of a phytophagous beetle, we review here the wealth of genetic and genomic information generated so far for the species-rich beetle superfamilies Chrysomeloidea (longhorn, seed and leaf beetles) and Curculionoidea (weevils and bark beetles). In this review we briefly describe the most common methods used to investi- gate the beetle genomes and also compile the nucleotide sequence information stored in public gene databases until December 2004. The motivations and relevance of these research initiatives are described in certain detail, distinguishing among structural and popu- lation studies, phylogenetic research, the study of genes involved in development and diapause, energetic metabolism, vegetal tissue digestion, and genes for insect resistance and defence.

[Background] Rates of molecular evolution are known to vary across taxa and among genes, and this requires rate calibration for each specific dataset based on external information. Calibration is sensitive to evolutionary model parameters, partitioning schemes and clock model. However, the way in which these and other analytical aspects affect both the rates and the resulting clade ages from calibrated phylogenies are not yet well understood. To investigate these aspects we have conducted calibration analyses for the genus Carabus (Coleoptera, Carabidae) on five mitochondrial and four nuclear DNA fragments with 7888 nt total length, testing different clock models and partitioning schemes to select the most suitable using Bayes Factors comparisons., [Results] We used these data to investigate the effect of ambiguous character and outgroup inclusion on both the rates of molecular evolution and the TMRCA of Carabus. We found considerable variation in rates of molecular evolution depending on the fragment studied (ranging from 5.02% in cob to 0.26% divergence/My in LSU-A), but also on analytical conditions. Alternative choices of clock model, partitioning scheme, treatment of ambiguous characters, and outgroup inclusion resulted in rate increments ranging from 28% (HUWE1) to 1000% (LSU-B and ITS2) and increments in the TMRCA of Carabus ranging from 8.4% (cox1-A) to 540% (ITS2). Results support an origin of the genus Carabus during the Oligocene in the Eurasian continent followed by a Miocene differentiation that originated all main extant lineages., [Conclusions] The combination of several genes is proposed as the best strategy to minimise both the idiosyncratic behaviors of individual markers and the effect of analytical aspects in rate and age estimations. Our results highlight the importance of estimating rates of molecular evolution for each specific dataset, selecting for optimal clock and partitioning models as well as other methodological issues potentially affecting rate estimation., This research was funded by projects CGL2006/06706, CGL2009-10906 (JS) and CGL2008-00007 (JGZ) of the Spanish Ministry of Science and Innovation (the latter with support of the European Regional Development Fund, "Una manera de hacer Europa"), as well as project 08724PI08 of the Fundación Séneca (Murcia) (JS). CA received the support of FPU predoctoral studentship of the Spanish Ministry of Education.