New descriptions of endophalli of Timarcha Samuelson, 1819 (Coleoptera, Chrysomelidae).

The endophalli of Timarcha arragonica Balbi, 1892, T. lusitanica (Fabricius, 1781), T. pratensis Duftschmid, 1825 and T. chloropus (Germar, 1823) are described and illustrated. The taxonomic relationships among these species and with those previously studied species are discussed.

The chiasmata systems of Scottish Chysolinalatecincta (Demaison, 1896) (Coleoptera, Chrysomelidae).

The meiotic systems of some Scottish individuals of the rare Chrysolinalatecinctassp.intermedia (Franz, 1938) have been analyzed from meiotic cells at diakinesis to study the types of chromosomal bivalents and the number and locations of their chiasmata. The mean number of unichiasmate was about two-thirds and that of bichiasmate bivalents about one-third. Most chiasmata were at distal positions and there were no pairwise statistically significant differences in the mean number of chiasmata and those of unichiasmate and bichiasmate bivalents between the three surveyed geographic sources of these Scottish individuals. However, pairwise significant differences were found in the mean number of proximal + interstitial chiasmata between Loch Etive (Argyllshire) and both Orkney and Shetland Islands individuals. The presumed higher values of genetic recombination due to the proximal + interstitial chiasmata with regard to the prevailing distal ones, might provide a slight selective advantage to the insular individuals against the more extreme climates of both islands compared with the Loch Etive site.

A cytogenetic analysis in two species of Cassidinae (Coleoptera, Chrysomelidae).

Two species of Cassidinae have been chromosomally analyzed, Cassida humeralis Kraatz, 1874 from France, with 2n = 18, 8 + Xyp meioformula and Anacassis fuscata (Klug, 1829) from Uruguay, with 2n = 30, 14 + Xy meioformula. The karyotype of the former is composed of similar meta/submetacentric autosomes, a small X-chromosome and a tiny y-chromosome, as many other Cassida and tribe Cassidini species, whereas that of the latter has four pairs of acro/telocentric autosomes at least and the remaining meta/submetacentrics including the X-chromosome and a tiny y-chromosome, which points out to its probable apomorphic origin by centric fissions, as found in some other species of the tribe Mesomphaliini.

Endophallus structure: a promising tool for cryptic species identification in Timarcha Samouelle, 1819 (Coleoptera: Chrysomelidae: Chrysomelinae).

Contrary to the subtle differences of habitus found between many species of Timarcha their internal sacs of male genitalia have shown a remarkable variation. Thirty-two Palaearctic taxa, mostly from the Iberian Peninsula, have been analyzed for this trait, which can be used for species diagnosis and also for establishing species groups of close relatedness in agreement mainly with genetic analyses. According with this trait, new synonymies and taxonomical changes are proposed: T. intermedia carmelenae Petitpierre, 2013 stat. nov., T. intermedia kiesenwetteri Kraatz, 1879 stat. nov., T. intermedia lugens Rosenhauer, 1856 stat. nov.; T. sinuatocollis monserratensis Bechyné, 1962 comb. nov.; T. piochardi Fairmaire, 1874 stat. nov.; T. tortosensis Bechyné, 1948 stat. nov.; T. perezii Fairmaire, 1884 syn. nov. and T. asturiensis Kraatz, 1879 syn. nov. = T. geniculata Germar, 1824. Furthermore, the endophalli of T. hummeli, T. carmelenae, T. kiesenwetteri, T. lugens, T. tenebricosa, T. parvicollis, T. insparsa, T. marginicollis, T. balearica, T. strangulata spp., T. calceata, T. scabripennis, T. espanoli, T. monticola, T. cyanescens, T. interstitialis, T. aurichalcea, T. oblongula, T. hispanica and T. granadensis are illustrated. One new species, T. aitanae sp. nov. is described.

Updated checklist of Balearic leaf beetles (Coleoptera: Chrysomelidae).

The first updated checklist of Balearic leaf beetles (Chrysomelidae) since 1960 is presented here, evincing the presence of 118 species. This estimation is clearly lower than the 141 species reported in the only list available to date (Jolivet, 1953), and the dissimilarity is even more pronounced if we take into account that 22 new species have been added during this period. The possible explanations for these differences are discussed. The main island in the archipelago holds most of the species (Mallorca, 113 spp.), followed by Menorca (71 spp.), Eivissa (39 spp.) and Formentera (19 spp.). Thus, the Gymnesian islands (Mallorca and Menorca) are more species-rich than the Pityusic ones (Eivissa and Formentera). The number of species per island is significantly correlated with their respective areas not only for the Balearic but also for the much larger western Mediterranean islands of Corsica and Sardinia, and these abundances are not related with their nearness to the closest mainland. Among the different subfamilies and tribes, the Balearic flea-beetles (Alticinae) are clearly more prevalent whereas on the contrary, the Clytrini are less represented in comparison with the nearest mainland (Iberian Peninsula). The presented checklist includes four endemic species, Cryptocephalus majoricensis (Mallorca, Menorca and Formentera), C. tramuntanae (Mallorca), Cyrtonus majoricensis (Mallorca) and Timarcha balearica (Mallorca and Menorca). Furthermore, two adventitious species, Monoxia obesula and Epitrix hirtipennis of North American origin, have been reported for the first time in the Balearic Islands, in agreement with previous findings in other Mediterranean countries.

A chromosomal analysis of three species of Timarcha (Coleoptera, Chrysomelidae, Chrysomelinae).

The karyotypes of three species of Timarcha Latreille, 1829 have been analysed. Timarcha (Metallotimarcha) metallica (Laicharting, 1781), has 18 + Xyp male meioformula and 2n = 38 chromosomes, similar to those found in the two species of subgenus Americanotimarcha Jolivet, 1948, in agreement with morphological and molecular phylogenetic grounds. Timarcha (Timarcha) carmelenae Petitpierre, 2013 displays 9 + Xyp and 2n = 20 chromosomes as in morphologically related Andalusian species, whereas Timarcha (Timarcha) parvicollis ssp. seidlitzi Kraatz, 1879 shows 11 + Xyp and 2n = 24 chromosomes, clearly differing from the previous species. These results are discussed in order to get an insight into the main trends of the chromosomal evolution in Timarcha.

New contributions to the molecular systematics and the evolution of host-plant associations in the genus Chrysolina (Coleoptera, Chrysomelidae, Chrysomelinae).

The taxonomic circumscription of the large and diverse leaf beetle genus Chrysolina Motschulsky is not clear, and its discrimination from the closely related genus Oreina Chevrolat has classically been controversial. In addition, the subgeneric arrangement of the species is unstable, and proposals segregating Chrysolina species into new genera have been recently suggested. In this context, the availability of a phylogenetic framework would provide the basis for a stable taxonomic system, but the existing phylogenies are based on few taxa and have low resolution. In the present study we perform a phylogenetic analysis based on mitochondrial (cox1 and rrnL) and nuclear (H3) DNA sequences from a sample of fifty-two Chrysolina species representing almost half of the subgeneric diversity of the group (thirty out of sixty-five subgenera) and most of the morphological, ecological and karyological variation in the genus. In addition, five Oreina species from two subgenera have also been analysed. The resulting phylogeny is used to evaluate some of the most relevant taxonomic hypotheses for Chrysolina, and also to reconstruct its ancestral host plant associations in a Bayesian framework. Our findings support the paraphyly of Chrysolina as currently defined due to the inclusion of Oreina, the monophyly of the Chrysolina (plus Oreina) species including the divergent Chrysolina (Polysticta) vigintimaculata (Clark, 1864), and enable inferences of deep-level evolutionary relationships among the studied subgenera. The plant family Lamiaceae is inferred as the ancestral host of the study group, whose evolution is characterized by continuous host-shifting among pre-existing host plant families. Some Chrysolina clades include mixtures of species with different levels of diet breadth, indicating that niche width has varied through time.

A chromosomal analysis of twelve species of the subfamily Chrysomelinae (Coleoptera, Chrysomelidae).

Twelve species of chrysomelines, all but one from the Palaearctic region, have been cytogenetically analyzed, mostly through their male meiotic metaphases I. Ambrostoma superbum has 2n = 40 (Xy(p)), Chrysolina colasi, Oreina fairmairiana and the Neotropical Platyphora spectabilis have 2n = 24 (Xy(p)), Chrysolina gebleri 2n = 26 (XY(p)), Colaspidema barbarum 2n = 28 (Xy(p)), Crosita altaica and C. rugulosa 2n = 30 (Xy(p)), Phratora polaris, Ph. vitellinae and Ph. vulgatissima 2n = 34 (Xy(p)), and the karyotype of Chrysolina marginata, consisting of 40 chromosomes, is also described. These results are discussed with those previously obtained in related genera and congeneric species, giving further support and extending the high chromosomal variability so far found in this subfamily of leaf beetles.

A chromosomal analysis of four species of Chilean Chrysomelinae (Coleoptera, Chrysomelidae).

Four species of Chilean leaf beetles in the subfamily Chrysomelinae have been cytogenetically analyzed, Blaptea elguetai Petitpierre, 2011, Henicotherus porteri Bréthes, 1929 and Jolivetia obscura (Philippi, 1864) show 2n = 28 chromosomes and a 13 + Xyp male meioformula, and Pataya nitida (Philippi, 1864) has the highest number of 2n = 38 chromosomes. The karyotype of Henicotherus porteri is made of mostly small meta/submetacentric chromosomes, and that of Jolivetia obscura displays striking procentric blocks of heterochromatin at pachytene autosomic bivalents using conventional staining. These findings are discussed in relation to previous cytogenetic data and current taxonomy of the subfamily.

Cytogenetics, cytotaxonomy and chromosomal evolution of Chrysomelinae revisited (Coleoptera, Chrysomelidae).

Nearly 260 taxa and chromosomal races of subfamily Chrysomelinae have been chromosomally analyzed showing a wide range of diploid numbers from 2n = 12 to 2n = 50, and four types of male sex-chromosome systems. with the parachute-like ones Xy(p) and XY(p) clearly prevailing (79.0%), but with the XO well represented too (19.75%). The modal haploid number for chrysomelines is n = 12 (34.2%) although it is not probably the presumed most plesiomorph for the whole subfamily, because in tribe Timarchini the modal number is n = 10 (53.6%) and in subtribe Chrysomelina n = 17 (65.7%). Some well sampled genera, such as Timarcha, Chrysolina and Cyrtonus, are variable in diploid numbers, whereas others, like Chrysomela, Paropsisterna, Oreina and Leptinotarsa, are conservative and these differences are discussed. The main shifts in the chromosomal evolution of Chrysomelinae seems to be centric fissions and pericentric inversions but other changes as centric fusions are also clearly demonstrated. The biarmed chromosome shape is the prevalent condition, as found in most Coleoptera, although a fair number of species hold a few uniarmed chromosomes at least. A significant negative correlation between the haploid numbers and the asymmetry in size of karyotypes (r = -0.74) has been found from a large sample of 63 checked species of ten different genera. Therefore, the increases in haploid number are generally associated with a higher karyotype symmetry.

DNA barcoding insect-host plant associations.

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 barcodes 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.

A cytogenetic study on three Chilean species of Chrysomelinae (Coleoptera, Chrysomelidae).

Three species of Chilean leaf beetles were chromosomally analyzed. The endemic Araucanomela wellingtonensis displays a male meioformula of 13 + Xyp with 2n = 28 chromosomes and an asymmetric karyotype with two large autosome pairs and 12 medium/small pairs of autosomes and sex-chromosomes, a diploid number which had not been found among the other species of the subtribe Paropsina sensu lato studied to date. Strichosa eburata presents a meioformula of 11 + Xyp, 2n = 24 chromosomes, as occurs in many species of chrysomelines belonging to different subtribes. Furthermore, Phaedon cyanopterum has a 16 + Xyp meioformula, that is 2n = 34 chromosomes, of small size mostly, also in agreement with the karyological findings obtained in all the other congeneric species so far examined. These cytogenetic data are discussed with respect to the previous ones in this subfamily and with other characters of taxonomic and evolutionary value.

Evolution of low-copy number and major satellite DNA sequences coexisting in two Pimelia species-groups (Coleoptera).

Satellite DNA sequence evolution has been studied in several insect species from the genus Pimelia (Tenebrionidae, Coleoptera). Low-copy number homologs of the previously characterized major satellite DNA from P. monticola (PMON) have been cloned and sequenced from six congeneric species belonging to two species groups: Ibero-Balearic and Moroccan. Sequence analysis of a sample of low-copy number repeats revealed two subfamilies, differing on average 17.5% due to randomly spread single point mutations. Each subfamily is specific for a group of taxa in congruence with their biogeography. Within each group, there is no significant species-specific clustering of the sequences. These results suggest that the two satellite subfamilies arose after the split of an ancestral lineage into the North African and Ibero-Balearic Pimelia species-groups, but before their subsequent radiation. Rate heterogeneity tests suggest that PMON sequences have evolved faster in the lineage leading to the Moroccan group. Comparison of sequence divergences between minor PMON and the previously characterized major PIM357 satellite obtained from the same taxa, points to similar evolutionary dynamics. Both sequences are evolving in parallel accumulating mutations in a gradual manner irrespectively of significant differences in abundance. These data show that copy number of the sequence families does not necessarily affect the sequence change dynamics of satellite repeats.

Higher-order organization and compartmentalization of satellite DNA PIM357 in species of the coleopteran genus Pimelia.

The PIM357 satellite DNA family is present in 26 Pimelia taxa (Tenebrionidae, Coleoptera) with endemic congeneric species from the Canary Islands showing higher interrepeat variability than continental ones. In this paper, we compare the repetitive DNA sequences of a Canarian species that has distinct subfamilies of repeat units, P. radula ascendens, with another without such subfamilies, P. sparsa sparsa. The chromosomal localization of the repeat units and the comparison of the variability of randomly cloned monomers to the one estimated by comparing repeat units from dimers and trimers suggest the absence of satellite subfamilies in P. sparsa sparsa. Hence, the repeat units of this species seem to be uniformly and randomly distributed throughout all chromosomes out of one chromosomal pair. On the contrary, P. radula ascendens shows four divergent subfamilies of repeat units supported by several diagnostic nucleotide substitutions. These subfamilies seem to form four distinct repeat units: monomer subfamily 1, monomer subfamily 4 and two higher-order units (dimer linking subfamily 1 and 4, and dimer linking subfamily 2 and 3). Moreover, monomers of subfamily 1 are present in three chromosomal pairs only. We discuss the effect of different potential factors acting in the concerted evolution and the genomic organization of stDNA sequences in these taxa.

Evolutionary dynamics of satellite DNA family PIM357 in species of the genus Pimelia (Tenebrionidae, Coleoptera).

A large number of repeats of a satellite DNA (stDNA) family have been cloned and sequenced from species and populations of the genus Pimelia (Tenebrionidae, Coleoptera). The beetles were collected in the Canary Islands, Morocco, the Iberian Peninsula, and the Balearic Islands in order to analyze the evolutionary forces and processes acting on abundant stDNAs conserved at the genus level. This repetitive family is composed of an abundant A-T-rich stDNA, with basic units of 357 bp. All the sequences obtained showed similarity to the 22 repeat units of the PIM357 stDNA family described previously for six Iberian Pimelia species (Pons et al. 1997 ). An analysis based on similarity shows the presence of three different groups of sequences clearly in accordance with their geographical origin. One is composed of satellite sequences from Iberian and Balearic species, a second group from the Moroccan taxa, whereas the third one is from the Pimelia species endemic to the Canary Islands. The latter group shows higher nucleotide diversities for their stDNA sequences and a lack of relationship between transition stages to fixation and sequence divergence. Phylogeographic data of Canarian Pimelia show that the PIM357 stDNA family has persisted for more than 8 Myr and could probably be traced to the origin of the lineage. The data suggest that distinct demographic and phylogenetic patterns related to the colonization of the volcanic Canarian island chain account for particular evolutionary dynamics of the repeat DNA family in this group.