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There are 96 endemic species of Eumolpinae (Coleoptera: Chrysomelidae) described from New Caledonia, but some estimates propose that the actual number could be at least twice this figure. Not surprisingly, when a particular species assemblage has been revised, the number of species in that group increases significantly. Here, we revise the New Caledonian endemic genus Taophila Heller, 1916, the best studied in this fauna and currently known to include eleven species, one in the subgenus Jolivetiana Gómez-Zurita & Cardoso, 2014, and ten in the nominal subgenus. The analysis of morphological differences in a large sample of Taophila and the validation of the resulting species hypotheses in an integrative fashion based on a phylogenetic analysis of partial mtDNA sequences (cox1 and rrnS) resulted in the addition of eleven more taxa. Taxonomic splits mainly reinterpreted the previous observation of mtDNA paraphyly affecting T. subsericea Heller, 1916, shown to represent a complex of species mostly distinguishable by diagnostic differences among females. The new species described are: T. bituberculata n. sp., T. carinata n. sp., T. dapportoi n. sp., T. davincii n. sp., T. draco n. sp., T. goa n. sp., T. hackae n. sp., T. samuelsoni n. sp., T. sideralis n. sp., T. taaluny n. sp. and T. wanati n. sp. These additions and the synonymy T. subsericea Heller = Stethotes mandjeliae Jolivet, Verma & Mille, 2010 n. syn., bring to 21 the total number of species in Taophila. Moreover, we also found the first evidence of mtDNA introgression between species of New Caledonian Eumolpinae, resulting from putative recent hybridization of T. subsericea and T. dapportoi where these species coexist. We describe a model incorporating the mtDNA genealogy of T. subsericea about the conditions that may have favored the secondary geographic encounter required for the hybridization of these species.

In this work, we have examined a large sample of Eumolpinae leaf beetles from Nicaragua and found 18 species reported for the first time in this country, including the new species Caryonoda funebris n. sp., which also represents a new genus record for Central America, and two genera of Typophorini not reported from Nicaragua so far: Paria LeConte, 1858 and Tijucana Bechyné, 1957. Apart from the description of the new species and taxonomic commentaries on each of the new country records, we also illustrate these species along with drawings of male genitalia and spermathecae when available to assist the interpretation of our taxonomic decisions in the future. We take the opportunity in this work to formalize the combination of Chrysodina cupriceps Lefèvre, 1877 as Chrysodinopsis cupriceps (Lefèvre) n. comb., Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This project was possible thanks to funds from Grant CGL2017-83324-P of the State Research Agency of the Spanish Ministry of Science and Innovation, with co-participation of the European Regional Development Fund of the European Commission, as well as Project BIOCON08 191/09 of the Fundación BBVA in their Program on Ecology and Conservation Biology., Introduction Material and methods Results and discussion References Acknowledgements Funding Author information Ethics declarations Additional information Rights and permissions About this article

Recent studies suggest that endosymbionts of herbivore insects can be horizontally transferred to other herbivores feeding on the same host plants, whereby the plant acts as an intermediate stage in the chain of transmission. If this mechanism operates, it is also expected that insect communities sharing the same host plant will have higher chances to share their endosymbionts. In this study, we use a high-throughput 16S rRNA metabarcoding approach to investigate the presence, diversity, and potential sharing of endosymbionts in several species of leaf beetles (Coleoptera: Chrysomelidae) of a local community specialized on an alder diet in North America. Rickettsia and Wolbachia were predominant in the sample, with strong evidence for each species having their own dominant infection, of either or both types of bacteria. However, all species shared a much lower proportion of a particular Wolbachia type, compatible with the same strain dominant in one of the species of leaf beetles. Crucially, the same 16S rRNA haplotype of Wolbachia was found on alder leaf extracts. The combined evidence and the absence of this strain in a syntopic species of leaf beetle feeding on a different host plant support the hypothesis that at least the initial stages of the mechanism that would allow horizontal transmission of endosymbionts across species feeding on the same plant is possible. The accessibility and characteristics of endosymbiont associations of this system make it suitable for deeper analyses of their diversity and transmission in natural conditions., This study was possible specifically thanks to funds made available by the Spanish Ministry of Economy (MINECO) through grant no. CGL2014-52937-P. Project 2017SGR991 (Generalitat de Catalunya) contributed toward preparation of the article and publication costs. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

The fauna of Eumolpinae of New Caledonia has been under intense scrutiny in over the last decade and numerous species are being described on a regular basis. In this study, we revise in detail the group of Colaspis laboulbenei Montrouzier, 1861, currently in the genus Dematochroma Baly, 1864, using a combined approach with DNA-based phylogenies and the study of morphology and species distributions. Several relevant systematic conclusions result from this revision: (1) recognition of the monophyletic group around Dematochroma laboulbenei; (2) recognition of an important morphological character, namely the presence or absence of a dorsal groove on pygidium, separating two narrowly parapatric lineages in this group; (3) reversal of a long-held synonymy with D. laboulbenei and reinstatement of Thasycles cordiformis Chapuis, 1874 stat. rev.; (4) restitution of Thasycles Chapuis, 1874 stat. rev. based on phylogenetic and taxonomic arguments to delimit this group of species; (5) transfer of Dematochroma fusca Jolivet, Verma & Mille, 2007, D. laboulbenei and Dematochroma panieensis Jolivet et al., 2007 to Thasycles; and (6) description of seven new species, including Thasycles castaneus Gómez-Zurita n. sp., Thasycles compactus Gómez-Zurita n. sp., Thasycles grandis Gómez-Zurita n. sp., Thasycles magnus Gómez-Zurita n. sp., Thasycles puncticollis Gómez-Zurita n. sp., Thasycles tenuis Gómez-Zurita n. sp., and Thasycles variegatus Gómez-Zurita n. sp. A species identification key and a brief discussion about the systematics and potential factors affecting the diversification and distribution of Thasycles are provided., This work was possible thanks to several sources of funding, including Grant CGL2017-83324-P of the Spanish Ministry of Science and Innovation, with support from the European Regional Development Fund, and also to the European Commision Synthesys+ project (grant no. BE-TAF-81)., Keywords 1. Introduction 2. Material and methods 2.1. Sampling 2.2. Acquisition of DNA sequence data 2.3. Phylogenetic analyses 2.4. Morphological analyses 3. Results and discussion 3.1. Phylogenetic relationships of the groups of interest 3.2. Restatement and limits of the genus Thasycles Chapuis, 1874 3.3. Redescription of Thasycles 3.4. Fixing the meaning of old names 3.5. Taxonomic treatment 3.6. Identification key to the species of Thasycles 3.7. Evolution of Thasycles and the classification of Eumolpinae Declaration of competing interest Acknowledgements Appendix A. Supplementary data References

The monophyletic group of species around Dematochroma pilosa Jolivet, Verma & Mille is identified in this work by combining information from mitochondrial DNA data and morphological features. A series of defining traits diagnosing this species assemblage from its closest phylogenetic relatives, including the genera Thasycles Chapuis and Atrichatus Sharp, is used to argue for its taxonomic separation and propose a new genus, named Dematotrichus gen. nov. Both Dematochroma pilosa and Montrouzierella hispida Jolivet, Verma & Mille are transferred to the new genus as D. pilosus (Jolivet, Verma & Mille) comb. nov. and D. hispidus (Jolivet, Verma & Mille) comb. nov., and 11 new species are described: D. capillaris sp. nov., D. capillosus sp. nov., D. comans sp. nov., D. crinitus sp. nov., D. comatulus sp. nov., D. hirtus sp. nov., D. hirsutus sp. nov., D. horridus sp. nov., D. pubescens sp. nov., D. setosus sp. nov. and D. villosus sp. nov. The work includes an identification key for all the species in the new genus., This work was funded by Grant CGL2017-83324-P of the Spanish Ministry of Science and Innovation, with support from the European Regional Development Fund., Introduction Materials and methods Collections, specimens and morphological studies DNA extraction, mtDNA markers, sequencing and phylogenetic analyses Tree-based species delimitation Results and discussion Mitochondrial phylogeny and tree-based species delimitation Description of a new genus of New Caledonian Eumolpinae Supplemental material Acknowledgements Additional information References

New Caledonia is an important biodiversity hotspot, where numerous plant and animal groups show high levels of species diversity and endemicity, while facing multiple threats to their habitats. Leaf beetles in the subfamily Eumolpinae illustrate this pattern, with species estimates higher than the number of described taxa and distribution ranges that are often consistent with microendemicity. In this study, we increase the knowledge of this group by focusing on the Taophila subgenus Lapita, known from three species but here expanded to eleven with eight new species: T. atlantis sp. nov., T. hermes sp. nov., T. kronos sp. nov., T. oceanica sp. nov., T. olympica sp. nov., T. ouranos sp. nov., T. riberai sp. nov., and T. tridentata sp. nov. Additionally, we infer the evolutionary history of the group using mtDNA markers (COI and rrnS). This phylogeny and the species distribution help hypothesize a model of evolution for this lineage in the context of historical climatic and geological changes of New Caledonia., This work was funded by the Spanish ‘Agencia Estatal de Investigación’ through Grant No. CGL2017-83324-P and the associated predoctoral scholarship PRE2018- 083172 to LP, both with support from the European Regional Development Fund of the European Commission.

In this work, the Chrysomelinae leaf beetle subgenus Calligrapha s. str. Chevrolat, 1836 is revised, offering redescriptions and keys for identification of twelve species currently considered in this group, allied to the South American species Calligrapha polyspila (Germar, 1821), the generic type of Calligrapha. The current species count results from important taxonomic changes. These include reversing a long-held synonymy, resurrecting the name Calligrapha mexicana Stål, 1859 stat. rev. for a species that is different from Chrysomela serpentina Rogers, 1856; upgrading the status of Polyspila serpentina var. discrepans Achard, 1923 to Calligrapha discrepans (Achard) stat. rev.; and formally proposing a number of new synonymies for several species, including: (1) Calligrapha discrepans (Achard) (= Calligrapha serpentina ssp. temaxensis Bechyné, 1952 syn. nov.); (2) Calligrapha fulvipes (Gistel, 1848) (= Calligrapha bajula Stål, 1860 syn. nov.; = Calligrapha nupta Stål, 1859 syn. nov.; = C. sponsa Stål, 1859 syn. nov.); and (3) Calligrapha polyspila (Germar) (= Polyspila polyspila var. bilineolata Achard, 1923 syn. nov.; = Polyspila polyspila var. plagata Achard, 1923 syn. nov.).

With 5,300 species, the Cryptocephalinae is the fourth largest subfamily of the megadiverse Chrysomelidae beetles. This subfamily currently merges groups that were traditionally considered subfamilies of their own, including the fulcidacines, clytrines and cryptocephalines, and it is believed to be sister to the Lamprosomatinae, forming a natural group characterized by immature stages developing within the protection of a solid case of organic matter known as scatoshell. However, hypotheses on the higher-level systematics of this diverse group and the recognition of its closest relatives have been numerous and none of them tested with phylogenetic approaches, save for an unpublished study based on morphology. In this work, we provide the first molecular phylogenetic hypothesis for a large dataset of Cryptocephalinae (167 species, 66 genera) based on the analysis of five gene fragments, including up to 3,488 aligned nucleotide positions of the mitochondrial DNA cox1 and rrnS, and the nuclearly encoded LSU, elongation factor 1-alpha and polyadenylate binding protein 1. The phylogeny confirms the sister-group relationship between Cryptocephalinae and Lamprosomatinae and identifies the Eumolpinae as the sister to this assemblage. Moreover, it partly refutes the current system for Cryptocephalinae giving support to five tribes in the subfamily: Fulcidacini and Clytrini, matching the current system, Pachybrachini separated from Cryptocephalini, and a new subantarctic tribe, Mylassini trib. n., divergent from the other lineages. Among Clytrini, data support inclusion of Ischyopachina within New World Babiina, which together with New World Megalostomina would be sister to Old World Clytrina. In Cryptocephalini, after exclusion of pachybrachines, four subtribes are distinguished, of which only Stylosomina matches the current system. Otherwise, the current Monachulina are dismantled into three clades deserving subtribal status, one of which, former New World Monachulina genera, is unambiguously transferred to Cryptocephalina. A molecular clock calibrated using paleontological and biogeographic information places the origin of the subfamily in the Early Cretaceous and the split of the main evolutionary lineages temporally and geographically congruent with the fragmentation of major Gondwanan territories., This work was funded by the “Agencia Estatal de Investigación” of the Spanish Ministry of Science and Innovation with Grant No. CGL2017-83324-P, also with the contribution of funds from the European Regional Development Fund, 1 INTRODUCTION 2 MATERIAL AND METHODS 2.1 Sampling 2.2 Phylogenetic marker selection and laboratory procedures 2.3 Sequence editing and preparation of phylogenetic matrices 2.4 Phylogenetic inference 2.5 Topology tests 2.6 Molecular clock analyses 3 RESULTS 3.1 Molecular dataset available for analysis 3.2 Phylogenetic relationships of Cryptocephalinae with other subfamilies 3.3 Molecular phylogeny of the Cryptocephalinae 3.4 Validation of monophyly hypotheses 3.5 Inferred ages of relevant clades 4 DISCUSSION 4.1 Phylogenetic support of Cryptocephalinae and their relatives 4.2 Higher-level systematics of the Cryptocephalinae 4.3 The tribes of Cryptocephalinae 4.3.1 Mylassini, a new tribe of Cryptocephalinae 4.3.2 Fulcidacini 4.3.3 Pachybrachini 4.3.4 Clytrini 4.3.5 Cryptocephalini 4.4 The evolution of the Cryptocephalinae ACKNOWLEDGEMENTS

In this work, two endemic genera of Eumolpinae from New Zealand, Pilacolaspis Sharp, 1886 and Peniticus Sharp, 1876, are revised for the first time since the description of their constituent species in the late 19th and early 20th centuries. The five taxa of Pilacolaspis are considered to comprise two species, P. huttoni (Broun, 1880) (=P. wakefieldi Sharp, 1886 n. syn. =P. rugiventris Broun, 1914 n. syn.) and P. latipennis Broun, 1912 (=P. angulatus Broun, 1912 n. syn.), with allopatric distributions in the Canterbury/Otago lowlands and the Marlborough areas around the Kaikouras, respectively. The following five species are recognised in Peniticus, one of which is new: P. antiquus Sharp, 1876, P. wallacei Broun, 1909, P. microreticulatus sp. nov., P. suffusus Sharp, 1876 (=P. robustus Sharp, 1880 n. syn.) and P. plicatus Broun, 1921. A key to the New Zealand genera of Eumolpinae and the species of Pilacolaspis and Peniticus is provided., This work would have not been possible without support from a Salvador de Madariaga mobility scholarship from the Spanish Ministry of Science, Innovation and Universities (PRX18/00219), as well as research grant CGL2017‐83324‐P of the Agencia Estatal de Investigación from the same Ministry, with contribution from the European Commission RDF.

A local survey of Chrysomelidae in a coastal area of some 10 km2 in the Núi Chúa National Park (Ninh Thuan, S Vietnam) yielded a remarkable number of Galerucinae. Most of them, up to 18 species, could be ascribed to the informal group Monoleptites, i.e. galerucines with very long first metatarsomere, longer than the remaining four combined. These representatives of Monoleptites constitute a strongly supported monophyletic group based on a combined analysis of one mtDNA (cox1) and one nuclear (wnt) marker, and include up to thirteen species which are not recognisable as species previously described or reported from Vietnam or neighbouring countries. These species are described herein, including eleven species tentatively assigned to the genus Monolepta Chevrolat (M. decretasp. nov., M. deminutasp. nov., M. densopunctatasp. nov., M. dubiasp. nov., M. fluctuanssp. nov., M. fuscicornesp. nov., M. interruptomarginatasp. nov., M. ochraceasp. nov., M. praetextasp. nov., M. quotidianasp. nov. and M. thomaswagnerisp. nov.) and two to the genus Paleosepharia Laboissière (P. frontissp. nov. and P. nuichuasp. nov.). Moreover, the name Desbordeleptanom. nov. is proposed to remove the homonymy of Desbordesius Laboissière, 1936 nec Maindron, 1906, and the spermatheca of D. picea (Kimoto) and male and female genitalia of M. dalatica Kimoto and M. semicostata Kimoto are described for the first time. Finally, a molecular analysis of putative diets based on the identification of chloroplastic psbA-trnH sequences obtained from total DNA extractions of the beetles, provides with some robust hypotheses for the diet of these animals, showing that they are polyphagous, and able to utilise a wide range of mainly eudicots in their environment. The results of this study roughly represent a 12% increase in the number of known species of the Monoleptites group in Vietnam and highlight the need for a profound revision of generic attributes in this group., This work was possible thanks to funds from the Spanish Ministry of Economy and Competitiveness to promote a Scientific Cooperation Agreement between the Spanish High Research Council (CSIC) and the Vietnamese Academy of Sciences and Technology (VAST).

Tricholapita Gómez-Zurita and Cardoso nom. nov. is proposed as the replacement name for the leaf beetle taxon Lapita Gómez-Zurita and Cardoso, 2014, nec Bickel, 2002. Moreover, the rank of Tricholapita stat. nov. is elevated from subgenus of Taophila Heller, 1916 to generic status. Phylogenetic evidence based on mtDNA rrnS sequences and diagnostic morphological characters reveals a new species from the south of Grande Terre in New Caledonia, which is described: Tricholapita reidi sp. nov.

Lamiinae is the most diverse subfamily of longhorned beetles, with about 20,000 described species classified into 80 tribes. Most of the tribes of Lamiinae were proposed during the 19th century and the suprageneric classification of the subfamily has never been assessed under phylogenetic criteria. In this study, we present the first tribal-level phylogeny of Lamiinae, inferred from 130 terminals (representing 46 tribes, prioritizing generic type species of the tribes) and fragments of two mitochondrial and three nuclear markers (cox1, rrnL, Wg, CPS and LSU; 5,024 aligned positions in total). Analyses were performed under Maximum Likelihood and Bayesian methods based on two datasets: a dataset including all taxa available for the study, and a reduced dataset with 111 terminals where taxa only contributing with mitochondrial markers were excluded from the matrix. The monophyly of Lamiinae was corroborated in three of the four analyses and 11 of the 35 tribes with more than one species represented in the analyses were consistently recovered as monophyletic. However, 15 tribes were not retrieved as monophyletic, requiring a revision of their boundaries: Acanthocinini, Acanthoderini, Agapanthiini, Apomecynini, Desmiphorini, Dorcaschematini, Enicodini, Hemilophini, Monochamini, Onciderini, Parmenini, Phytoeciini, Pogonocherini, Pteropliini and Saperdini. Based on these results, when strong support values for paraphyly were recovered, we argue a number of tribe synonymies, including Moneilemini as synonym of Acanthocinini; Onocephalini of Onciderini; Dorcadionini, Gnomini, Monochamini and Rhodopinini of Lamiini; and Obereini and Phytoeciini of Saperdini. Other taxonomic changes proposed in this study based on the criterion of monophyly and supported by morphological characters include the transfer of Tricondyloides and Stenellipsis to Enicodini, and of Dylobolus stat. rest., which is removed as subgenus of Mecas and restituted as genus, to Hemilophini. Furthermore, our analyses suggest that Ostedes and Neohoplonotus should be removed from Acanthocinini and Parmenini, respectively, and Colobotheini should be redefined to encompass several genera currently placed in Acanthocinini., This work was supported by the International Cooperation Programme iCOOP+ 2014 of the Consejo Superior de Investigaciones Científicas (Spain) [grant number COOPA20068]; the Conselho Nacional de Desenvolvimento Científico e Tecnológico (Brazil) [process numbers 234424/2014-1, 308994/2018-3, 304318/2018-3]; and the Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (Brazil) [process numbers E-26/202.381/2017, E-26/202.382/2017].

The leaf beetle genus Calligrapha is one of the few examples of animals with several obligate unisexual, female‐only species. Previous work showed that each one arose independently from interspecific hybridization events involving different species. However, all of them clustered in a single mtDNA clade together with some individuals of the parental bisexual species, which appeared as deeply polyphyletic in the mtDNA genealogy of the genus. The dating of these splits using a molecular clock placed them in the Quaternary and it was hypothesized that climatic change during this period may have favored range expansions and secondary contacts required for hybridization. In this work, we test this hypothesis and the origins of unisexuality in Calligrapha examining the diversity of mitochondrial (cox1) and nuclear (wingless, Wg) genes and the Bayesian continuous mtDNA phylogeography of a sample of more than 500 specimens of two bisexual species of Calligrapha at a continental scale and two unisexual species derived from them. Besides a major topological difference, whereby each bisexual species is monophyletic for Wg but paraphyletic for cox1, both gene datasets are consistent with a minimum of seven evolutionary lineages, coherent with geography and consistent with an ordered expansion to occupy their current ranges. The results also imply their survival in well‐established glacial refuges during the Last Glacial Maximum (LGM). Thus, for bisexual C. multipunctata there are two main, southern and northern lineages. The southern lineage expanded its range in two evolutionary branches, to the Rocky Mountains and to the northern Mississippi and Ohio River basins, respectively. The northern lineage has one branch in the Upper Mississippi and one that expanded west to the Pacific Northwest and east to the northeastern North Atlantic, finding refuge in both areas. These major lineages are parapatric in the Northern Great Plains, an area consistent with them having found refuge in the so‐called Driftless region during the LGM. For bisexual C. philadelphica, one northern lineage expanded west from the northern Appalachians and one east and southwest along the axis of the Appalachians, and the timing of events is consistent with their persistence in glacial refugia at both sides of the main Great Lakes lobe of the Laurentide Ice Sheet. There is evidence that the northeastern North Atlantic lineages of both species hybridized at the edge of their ranges after the LGM. The additional, divergent mtDNA lineage of these species shows evidence of range expansions of two lineages, one for each species, in an area south of the Laurentide Ice Sheet, and giving origin to the unisexual species by way of hybridization with other species in the Alleghanian region after the LGM. Interestingly, the individuals of supposedly bisexual species in this clade are all females. This suggests that unisexuality actually predates the origin of unisexual taxa in this system and that some bisexual species in Calligrapha may be species complexes instead, with cryptic species differing in their reproductive mode., This research was possible specifically thanks to funds made available by the Spanish Ministry of Economy (MINECO) through Grant No. CGL2014‐52937‐P, but it also benefited from samples and knowledge acquired thanks to previous projects funded by the same Organism, including Grant Nos. CGL2011‐23820 and CGL2008‐00007, the latter with contribution from the European Union Regional Development Fund (ERDF).

The interactions between herbivores and their host plants play a key role in ecological processes. Understanding the width and nature of these interactions is fundamental to ecology and conservation. Recent research on DNA-based inference of trophic associations suggests that the host range of phytophagous insects in the tropics may be wider than previously thought based on traditional observation. However, the reliability of molecular inference of ecological associations, still strongly dependent on PCR and thus exposed to the risk of contamination with environmental DNA, is under debate. Here, we explored alternative procedures to reduce the chance of amplification of external, nondiet DNA, including surface decontamination and analysis of mid/hind guts, comparing the results with those obtained using the standard protocol. We studied 261 specimens in eight species of Neotropical Chrysomelidae that yielded 316 psbA-trnH intergenic spacer sequences (cpDNA marker of putative diets) from unique and multiple-band PCR results. The taxonomic identity of these sequences was inferred using the automated pipeline BAGpipe, yielding results consistent with 31 plant families. Regardless of the protocol used, a wide taxonomic spectrum of food was inferred for all chrysomelid species. Canonical Correspondence Analysis using these data revealed significant differences attributed mainly to species (expectedly, since they represent different ecologies), but also to treatment (untreated vs. cleaned/gut samples) and PCR results (single vs. multiple bands). Molecular identification of diets is not straightforward and, regardless of the species' niche breadth, combining approaches that reduce external contamination and studying multiple individuals per species may help increasing confidence in results.

The Eumolpinae (Coleoptera: Chrysomelidae) from New Zealand are poorly known in terms of species diversity and, logically, of any downstream understanding of species features, including species distribution or ecology. This is true even though some of the species have been recognised as agricultural pests and associated with economic losses in the country. In this work, I have analysed the diversity and distribution of two genera of Eumolpinae that had been the subject of a taxonomic revision in the 1950s, Atrichatus Sharp and Eucolaspis Sharp. My observations, based on relatively abundant material from three collections and newly collected specimens, generally agree with the conclusions of that work, identifying two species of Atrichatus and at least five of Eucolaspis. I provide a new taxonomic character, namely the description of the spermathecae of all the species, as well as refined distribution maps based on available collection data. One new species of Eucolaspis is described, E. kotatousp. nov., from Te Paki, near Cape Reinga (Northland). In addition, E. antennata Shaw, previously known from only two specimens without locality data, is reported from Rotorua (Bay of Plenty). Despite loose similarities, Atrichatus–mainly distributed in the north of the South Island–and Eucolaspis–broadly sympatric with Atrichatus, but more diverse and widely distributed in the North Island–should not be considered as close relatives based on an important diagnostic character for higher-level systematics of Eumolpinae, namely a dorsal groove on the pygidium, present in the former and lacking in the latter. http://www.zoobank.org/urn:lsid:zoobank.org:act:05CCCC7C-E8DE-41F3-B0D1-F2090D79754B.

In this work, I revise a group of thirteen species in a natural assemblage of Calligrapha Chevrolat previously shown to represent a distinct and highly divergent lineage relative to other Calligrapha. The group is given subgeneric status, under the name Erythrographa subgen. nov., based on the morphological features of its representatives, which include reddish testaceous color to dark parts of body, including elytral markings, the presence of two spots enclosed by the humeral lunule, and a bifid end of the flagellum in male genitalia, among other typical features. One of the species of the new subgenus is also new and formally described as Calligrapha synthesys sp. nov. All the species of the subgenus Erythrographa subgen. nov. are found in Mexico (with the exception of C. wickhami, only known from southern Texas), with five species with larger distributions, reaching Nicaragua (C. notatipennis Stål), Costa Rica (C. labyrinthica Stål) or Panama (C. suboculata Stål, C. synthesys sp. nov. and C. tortilis Stål). The subgenus can be considered Neotropical, endemic of Central America and particularly diverse in the Mexican Transition Zone, between the Nearctic and Neotropical realms.

In this article, I describe a new endemic genus of New Caledonian Eumolpinae, Kumatoeides gen. nov. (tribe Eumolpini), and eight new species currently conforming the genus: K. anomala sp. nov., K. aulacia sp. nov. (generic type), K. leptalei sp. nov., K. megale sp. nov., K. metallica sp. nov., K. millei sp. nov., K. tarsalis sp. nov. and K. wanati sp. nov. Some easily recognizable defining traits of this new genus of fairly small species (2.23–3.20 mm) are the glabrous dorsum, aligned punctures on elytra in eight regular striae plus short scutellar and a subhumeral striae, convex elytral intervals, and the enlarged and elongate first pro- and mesotarsomeres of males. One species originally described in the genus Montrouzierella Jolivet, Verma et Mille is transferred to the new genus as Kumatoeides costata (Jolivet, Verma et Mille) nov. comb. A preliminary phylogenetic analysis using partial cox1 and rrnS mtDNA sequences of five species of Kumatoeides gen. nov. together with a representative sample of species of Eumolpinae from New Caledonia supports the monophyly of the genus and the existence of two main groups as recognized based on morphological traits. However, data are not conclusive about the relationship of the new genus with other New Caledonian lineages, although suggest a closest relationship with some unidentified species most similar to some representatives of the genus Samuelsonia Jolivet, Verma et Mille but with morphological similarities with Kumatoeides gen. nov., including dilated male protarsomeres, similar antennomere proportions and tendency to aligned rows of punctures on elytra, at least apically.

The digitization of museum specimens is a key priority in the Digital Era. Digital databases help to avoid unnecessary manipulation hazards to delicate collections, increase their accessibility to third party researchers, and contribute to the ongoing documentation of global biodiversity. Time, workforce and the need of specialized infrastructures limit the processing of the vast number of specimens in natural history collections. Cheaper, easy-to-use methods and volunteer programs are developing quickly to help bridge the gap. We present the results of combining citizen science for the digitization of an entomological collection in conjunction with the cooperation of a taxonomic expert for the remote identification of samples. In addition, we provide an assessment of the avoided monetary costs and the time needed for each step of the process. A photographic inventory of specimens belonging to the leaf beetle genus Calligrapha was compiled by volunteers using a low-cost compact camera and the species were identified using these images. Using digital photographs allowed for a rapid screening of specimens in the collection and resulted in an updated taxonomic identification of the Calligrapha collection at the Royal Belgian Institute of Natural Sciences. The pictures of the specimens and their original labels, as well as the new information from this endeavor were placed in an online public catalogue. This study demonstrates a worked example of how digitization has led to a practical, useful outcome through cooperation with an end user and highlights the value of museum collection digitization projects.

We report the architecture of testis transcriptomes of four closely related species of Calligrapha (Chrysomelidae) beetles, which diverged during the last 3 million years. Five cDNA libraries were sequenced using Illumina HiSeq technology, retrieving 102 884–176 514 assembled contigs, of which ~33–45% of these longer than 499 nt were functionally annotated. Annotation and sequence similarity comparisons of these libraries revealed high homogeneity in gene composition and the presence of several functional candidates related to reproduction or reproductive processes (0.72–1.08% of annotated sequences). Stringent sequence similarity analyses of these transcriptomes against empirically demonstrated male-biased genes in Drosophila melanogaster and Tribolium castaneum allowed the identification of 77 homologues in Calligrapha, possible candidates of male-biased expression. Some of these genes – including CG9313, Tektin-A or tomboy40 – were confirmed as orthologs of these male-biased genes using phylogenetic inference and available model insect data, increasing our confidence that they represent functional homologues too. Our transcriptomes are a valuable transcriptomic resource for the analysis of male-biased genes in Calligrapha, which has the added interest of including several female-only species. But it simultaneously represents a landmark for similar studies in Coleoptera, broadening the taxonomic diversity currently represented by the model species T. castaneum, and incipient genomic data in other herbivorous lineages, including weevils, longhorn beetles and leaf beetles., This research was funded by project CGL2011-23820 of the Spanish Ministry of Economy and Competitivity to JGZ, and an associated predoctoral studentship of the FPI programme to HIVR.

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.

Calligrapha (Coleoptera: Chrysomelidae) is a genus with species present in most of the American continent, from the Arctic polar circle to the Pampas in Argentina. In its current concept, the genus comprises some 80 species, but the diagnosis of the genus is problematic, based on a combination of potentially symplesiomorphic character states. In this study, we investigate the largest taxonomic sample of Calligrapha diversity to date (43 species) using a phylogenetic perspective based on more than 6000 molecular characters from eight genes (four mitochondrial and four nuclear) for a systematic evaluation of the genus. The analyses also include thirteen species in the closely related Zygospila (currently a subgenus of Zygogramma) to assist the systematic delimitation of Calligrapha. Partitioned and total evidence phylogenetic trees were additionally used for molecular clock analyses and dating based on standard mtDNA evolutionary rates, and for likelihood-based inference of ancestral areas. Calligrapha and Zygospila are reciprocally paraphyletic, and our interpretation of taxonomic stability merges both taxa into a larger genus Calligrapha which plausibly originated in the dry steppes of southern North America in the Late Miocene. The genus includes a minimum of five strongly supported lineages which initially diversified in the Pliocene, fully congruent with expectations from morphology, but of uncertain mutual relationships. Only two of these lineages dispersed to South America: the group of C. polyspila right at the time of the final closure of the Isthmus of Panama in the Early Pliocene and the group of C. argus only in recent times, well in the Pleistocene. The most species-rich lineage of Calligrapha, associated to trees and shrubs typical of riverine and lacustrine environments (as opposed to herbaceous steppe plants, generally Malvaceae and Asteraceae, for most other groups) diversified and spread in North America in the Late Pliocene. The ecological shift to a stable habitat spreading in the continent due to climate change is hypothesized as one possible explanation for the evolutionary success of this group. © 2014 The Norwegian Academy of Science, This study was possible primarily thanks to the project CGL2008-00007/BOS of the Spanish Ministry of Science and Innovation (with support from the EU FEDER Fund) to JGZ and an FPI scholarship associated to this same project to TM. This work also benefited from several grants of the EU Synthesys Programme (SE-TAF-1893, GB-TAF-4296 and DE-TAF-4348).

In species differentiation, characters may not diverge synchronously, and there are also processes that shuffle character states in lineages descendant from a common ancestor. Species are thus expected to show some degree of incongruence among characters; therefore, taxonomic delimitation can benefit from integrative approaches and objective strategies that account for character conflict. We illustrate the potential of exploiting conflict for species delimitation in a study case of ground beetles of the subgenus Carabus (Mesocarabus), where traditional taxonomy does not accurately delimit species. The molecular phylogenies of four mitochondrial and three nuclear genes, cladistic analysis of the aedeagus, ecological niche divergence and morphometry of pronotal shape in more than 500 specimens of Mesocarabus show that these characters are not fully congruent. For these data, a three-step operational strategy is proposed for species delimitation by (i) delineating candidate species based on the integration of incongruence among conclusive lines of evidence, (ii) corroborating candidate species with inconclusive lines of evidence and (iii) refining a final species proposal based on an integrated characterization of candidate species based on the evolutionary analysis of incongruence. This procedure provided a general understanding of the reticulate process of hybridization and introgression acting on Mesocarabus and generated the hypothesis of seven Mesocarabus species, including two putative hybrid lineages. Our work emphasizes the importance of incorporating critical analyses of character and phylogenetic conflict to infer both the evolutionary history and species boundaries through an integrative taxonomic approach. © 2014 John Wiley & Sons Ltd., This research was funded by projects of the Spanish Ministry of Science and Innovation CGL2006/06706, CGL2009-10906 (JS) and CGL2008-00007 with cofunding by European Union FEDER Funds (JGZ), as well as project 08724PI08 of the Fundación Séneca (Murcia) (JS). CA and PA received the support of two FPU predoctoral studentships of the Spanish Ministry of Education.

This study was made possible thanks to funds provided by the Committee for Research and Exploration of the National Geographic Society (Grant No. 8380-07), and a travel grant from the Percy Sladen Memorial Fund of the Linnean Society. The work benefited also from support from the Fundacion BBVA.

This study was possible thanks to funds provided by the Committee for Research and Exploration of the National Geographic Society (grant no. 8380-07), and a travel grant from the Percy Sladen Memorial Fund of the Linnean Society. The work benefited also from the support of the Juan de la Cierva Programme (Spanish Ministry of Science, Technology and Innovation) to the first author, and the Fundacion BBVA to the other authors. The Spanish National Institute for Bioinformatics and the IBE's IT team helped in the set-up for parallelization of processes. Fieldwork was carried out under the special authorization n° 6024-626 2008/DENV/SMT from the Direction de l'Environment (Noumea, New Caledonia) to our host in New Caledonia.

This research was funded by projects of the Spanish Ministry of Science and Innovation CGL2006/06706, CGL2009-10906 (JS), and CGL2008-00007, with co-funding by European Union FEDER Funds (J.G-Z.), as well as project 08724PI08 of the Fundacion Seneca (Murcia) (J.S.). C.A. received the support of an FPU predoctoral studentship of the Spanish Ministry of Education.

Integrative taxonomy is a recently developed approach that uses multiple lines of evidence such as molecular, morphological, ecological and geographical data to test species limits, and it stands as one of the most promising approaches to species delimitation in taxonomically difficult groups. The Pnigalio soemius complex (Hymenoptera: Eulophidae) represents an interesting taxonomical and ecological study case, as it is characterized by a lack of informative morphological characters, deep mitochondrial divergence, and is susceptible to infection by parthenogenesis-inducing Rickettsia. We tested the effectiveness of an integrative taxonomy approach in delimiting species within the P. soemius complex. We analysed two molecular markers (COI and ITS2) using different methods, performed multivariate analysis on morphometric data and exploited ecological data such as host–plant system associations, geographical separation, and the prevalence, type and effects of endosymbiont infection. The challenge of resolving different levels of resolution in the data was met by setting up a formal procedure of data integration within and between conflicting independent lines of evidence. An iterative corroboration process of multiple sources of data eventually indicated the existence of several cryptic species that can be treated as stable taxonomic hypotheses. Furthermore, the integrative approach confirmed a trend towards host specificity within the presumed polyphagous P. soemius and suggested that Rickettsia could have played a major role in the reproductive isolation and genetic diversification of at least two species.

This work was initiated under the sponsorship of the Alexander von Humboldt Fellowship to JGZ, and it received funding from project BA 2152/5-1 of the Deutsche Forschungsgemeinschaft (DFG) and from project CGL2008-00007 of the Spanish Ministry of Science and Innovation.

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.

New Caledonia is among Earth's biodiversity hotspots, and we are far from knowing how many species it sustains. We applied DNA-based methods for quick biodiversity assessment of New Caledonian Arsipoda flea beetles, enhancing the discovery and description of new species. Mitochondrial DNA phylogenetic analysis (cox1, rrnS) for four out of five known neocaledonian taxa hints at the existence of additional species, and two are confirmed and described based on morphology: Arsipoda geographica Gómez-Zurita sp. nov. and Arsipoda rostrata Gómez-Zurita sp. nov. Timing this small radiation using standard insect mitochondrial substitution rates places its origin in the Miocene. A DNA-based approach to investigate potential food plants for these herbivorous insects reveals associations with Myrsinaceae and Ericaceae, which have not yet been found in New Caledonia, suggesting that this indirect methodology may help in discovering undetected flora. Traditional taxonomy and molecular approaches cooperate here, boosting our knowledge on species inventory and ecological interactions where it is most needed. © 2010 Taylor & Francis., This project was funded by a Research Grant (no. 8380-07) of the Committee of Research and Exploration of the National Geographic Society, and also benefited from a travel grant from the Percy Sladen Memorial Fund of the Linnean Society of London.

A quantitative ecomorphological study was carried out on the sagitta otoliths of Antarctic and Subantarctic nototheniids, a particularly interesting family due to its fast adaptive radiation into different life strategies. We analyzed the otolith shape and size of 18 nototheniids to test the relationship between phylogeny, otolith characters and trophic niche. Relative size (area, length, width, perimeter and weight) and shape (biometric warp analysis based on homologous and pseudo-homologous landmarks) measurements were compared with phylogenic, habitat dwelling and food composition analyses. The results of the multivariate analysis of these factors indicate that there is a weak relationship between otolith shape of nototheniids and phylogeny; however, there is a clear correspondence between relative otolith size and shape and their trophic niche. The most benthic feeders of the family had the largest sagittae in relation to body size, and pelagic species had smaller and rounder shaped sagittae than benthic species. Consequently, in ecomorphological studies, it is useful to analyze the size and shape of sagittae. The discoidal shape of pelagic species, such as Pleuragrama antracticum, can be considered as a paedomorphic characteristic, converging to the juvenile sagittae of many species, which exhibit round sagittae that become elongated as they grow., This work was undertaken as part of the SCAR EASIZ program, and was supported by the Alfred Wegener Institute and the Spanish Antarctic Program (ANT1995-1249-E, ANT1997-1884-E, REN2000-1783-E).

Basal relationships in the Chrysomelidae (leaf beetles) were investigated using two nuclear (small and partial large subunits) and mitochondrial (partial large subunit) rRNA (≈ 3000 bp total) for 167 taxa covering most major lineages and relevant outgroups. Separate and combined data analyses were performed under parsimony and model-based tree building algorithms from dynamic (direct optimization) and static (Clustal and BLAST) sequence alignments. The performance of methods differed widely and recovery of well established nodes was erratic, in particular when using single gene partitions, but showed a slight advantage for Bayesian inferences and one of the fast likelihood algorithms (PHYML) over others. Direct optimization greatly gained from simultaneous analysis and provided a valuable hypothesis of chrysomelid relationships. The BLAST-based alignment, which removes poorly aligned sequence segments, in combination with likelihood and Bayesian analyses, resulted in highly defensible trees obtained in much shorter time than direct optimization, and hence is a viable alternative when data sets grow. The main taxonomic findings include the recognition of three major lineages of Chrysomelidae, including a basal “sagrine” clade (Criocerinae, Donaciinae, Bruchinae), which was sister to the “eumolpine” (Spilopyrinae, Eumolpinae, Cryptocephalinae, Cassidinae) plus “chrysomeline” (Chrysomelinae, Galerucinae) clades. The analyses support a broad definition of subfamilies (i.e., merging previously separated subfamilies) in the case of Cassidinae (cassidines + hispines) and Cryptocephalinae (chlamisines + cryptocephalines + clytrines), whereas two subfamilies, Chrysomelinae and Eumolpinae, were paraphyletic. The surprising separation of monocot feeding Cassidinae (associated with the eumolpine clade) from the other major monocot feeding groups in the sagrine clade was well supported. The study highlights the need for thorough taxon sampling, and reveals that morphological data affected by convergence had a great impact when combined with molecular data in previous phylogenetic analyses of Chrysomelidae. © The Willi Hennig Society 2007.

In this work, a group of seven species of Calligrapha (Coleoptera: Chrysomelidae) with pale spots contrasting with dark background of elytra is revised and their distributions specified based on the material studied from sixteen museums. Among these species, two are new and formally described: C. pavimentata sp. nov. and C. zapoteca sp. nov. A pragmatic identification key is provided to identify these taxa and it is proposed that these species belong to four different evolutionary lineages.

Trabajo presentado en el V Congreso de la Sociedad Espanola de Biologia Evolutiva (SESBE 2016), celebrado en Murcia del 18 al 21 de enero de 2016.

Our perception of diversity, including both alpha- and beta-diversity components, depends on spatial scale. Studies of spatial variation of the latter are just starting, with a paucity of research on beta-diversity patterns at smaller scales. Understanding these patterns and the processes shaping the distribution of diversity is critical to describe this diversity, but it is paramount in conservation too. Here, we investigate the diversity and structure of a tropical community of herbivorous beetles at a reduced local scale of some 10 km2, evaluating the effect of a small, gradual ecological change on this structure. We sampled leaf beetles in the Núi Chúa National Park (S Vietnam), studying changes in alpha- and beta-diversity across an elevation gradient up to 500 m, encompassing the ecotone between critically endangered lowland dry deciduous forest and mixed evergreen forest at higher elevations. Leaf beetle diversity was assessed using several molecular tree-based species delimitation approaches (with mtDNA cox1 data), species richness using rarefaction and incidence-based diversity indexes, and beta-diversity was investigated decomposing the contribution of species turnover and nestedness. We documented 155 species in the area explored and species-richness estimates 1.5–2.0x higher. Species diversity was similar in both forest types and changes in alpha-diversity along the elevation gradient showed an expected local increase of diversity in the ecotone. Beta-diversity was high among forest paths (average Sørensen's dissimilarity = 0.694) and, tentatively fixing at 300 m the boundary between otherwise continuous biomes, demonstrated similarly high beta-diversity (Sørensen's dissimilarity = 0.581), with samples clustering according to biome/elevation. Highly relevant considering the local scale of the study, beta-diversity had a high contribution of species replacement among locales (54.8%) and between biomes (79.6%), suggesting environmental heterogeneity as the dominant force shaping diversity at such small scale, directly and indirectly on the plant communities. Protection actions in the Park, especially these addressed at the imperative conservation of dry forest, must ponder the small scale at which processes shape species diversity and community structure for inconspicuous, yet extraordinarily diverse organisms such as the leaf beetles., This work results from the activity in the “Grup de Recerca Consolidad en Biologia Animal i Evolució” (project 2014SGR1532) and was possible thanks to funds from the Spanish Ministry of Economy and Competitiveness to promote a Scientific Cooperation Agreement between the Spanish High Research Council (CSIC) and the Vietnamese Academy of Sciences and Technology (VAST).

Beetles represent almost one-fourth of all described species, and knowledge about their relationships and evolution adds to our understanding of biodiversity. We performed a comprehensive phylogenetic analysis of Coleoptera inferred from three genes and nearly 1900 species, representing more than 80% of the world's recognized beetle families. We defined basal relationships in the Polyphaga supergroup, which contains over 300,000 species, and established five families as the earliest branching lineages. By dating the phylogeny, we found that the success of beetles is explained neither by exceptional net diversification rates nor by a predominant role of herbivory and the Cretaceous rise of angiosperms. Instead, the pre-Cretaceous origin of more than 100 present-day lineages suggests that beetle species richness is due to high survival of lineages and sustained diversification in a variety of niches.

The interactions between herbivores and their host plants play a key role in ecological processes. Understanding the width and nature of these interactions is fundamental to ecology and conservation. Recent research on DNA-based inference of trophic associations suggests that the host range of phytophagous insects in the tropics may be wider than previously thought based on traditional observation. However, the reliability of molecular inference of ecological associations, still strongly dependent on PCR and thus exposed to the risk of contamination with environmental DNA, is under debate. Here, we explored alternative procedures to reduce the chance of amplification of external, nondiet DNA, including surface decontamination and analysis of mid/hind guts, comparing the results with those obtained using the standard protocol. We studied 261 specimens in eight species of Neotropical Chrysomelidae that yielded 316 psbA-trnH intergenic spacer sequences (cpDNA marker of putative diets) from unique and multiple-band PCR results. The taxonomic identity of these sequences was inferred using the automated pipeline BAGpipe, yielding results consistent with 31 plant families. Regardless of the protocol used, a wide taxonomic spectrum of food was inferred for all chrysomelid species. Canonical Correspondence Analysis using these data revealed significant differences attributed mainly to species (expectedly, since they represent different ecologies), but also to treatment (untreated vs. cleaned/gut samples) and PCR results (single vs. multiple bands). Molecular identification of diets is not straightforward and, regardless of the species' niche breadth, combining approaches that reduce external contamination and studying multiple individuals per species may help increasing confidence in results.

Saline inland waters are globally threatened habitats harbouring many specialised endemic species, which often have restricted geographic ranges, and occur as highly isolated populations. We studied the genetic variation and phylogeography of Ochthebius glaber Montes and Soler, a rare and endangered water beetle endemic to hypersaline streams in the South and Southeast of the Iberian Peninsula. We used a 633 bp fragment of cytochrome oxidase subunit 1 gene to determine the genetic diversity and phylogeographic structure within this species, and interpret this in the light of the speciesȁ9 conservation requirements. Thirteen populations were sampled across the speciesȁ9 geographic range, and genetic diversity found to be very high, with 37 haplotypes across the 71 specimens examined (p-distance 0.2–7.3%, average 3.1±0.4). Phylogeographic analyses revealed a surprisingly high degree of geographical structure, detectable among populations separated by relatively short geographical distances, with three main groups of haplotypes which have apparently been isolated for significant periods of time. Past fragmentation and contiguous range expansion events were inferred as the main causes of the detected geographical associations of haplotypes. The establishment of independent evolutionary lineages as conservation units is particularly important for species inhabiting saline habitats such as O. glaber, which is endangered by habitat loss across most of its distribution. However, given the natural instability of hypersaline environments, the conservation of a network of populations and potential habitats would be necessary to enable the preservation of the process generating and maintaining the diversity of the species.

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.

We explore the correlational patterns of diet and phylogeny on the shape of the premaxilla and anterior tooth in sparid fishes (Perciformes: Sparidae) from the western Mediterranean Sea. The premaxilla is less variable, and in spite of the presence of species-specific features, a common structural pattern is easily recognizable in all species (i.e. the ascending and the articular processes are fused in a single branch, as in many percoid fishes). In contrast, tooth shape is more variable, and different structural types can be recognized (e.g. caninelike or incisive). Coupling geometric morphometric and comparative methods we found that the relationship between shape, diet and phylogeny also differs between premaxilla and tooth. Thus, the shape of the premaxilla is significantly correlated with food type, whereas the shape of the teeth is not correlated with diet, and probably reflects the species phylogenetic relationships. Two biological roles, resistance against compressive forces generated in the buccal cavity and the size of the oral gape, would explain the ecomorphological patterns of the premaxilla. The premaxilla and anterior tooth appear to evolve at different rates (mosaic evolution) and represent an example of morphological traits belonging to the same functional unit but following uncoupled evolutionary pathways.

Sixteen species of European cassidines, all but one of the genus Cussida, have been cytogenetically investigated, twelve of them for the first time. The diploid number of 18 chromosomes and 8 + Xy, meioformula is the prevalent karyotype of these species. The DNA content of Feulgen-stained spermatids shows a moderate variation, with most species having 0.8-1.1 pg. The possible trends of the chromosomal evolution in Cassida are discussed in the light of the present subgeneric taxonomy, host-plant selection, and other characteristics. The value of cytogenetics for the recognition and distinguishing between sibling species of Cassidu is also dealt with. Eduurd Petitpierre, Laborutori de Genitica, Depurtament de Biologia Ambientul, Fucultut de Ci&cies, UIB, 07071 Palma de Mullorca, Espaiia

Molecular genetic analyses of populations showing phenotypic variation can provide important insights into the nature and limits of species. This is especially true when interpopulation variation is assessed in a phylogenetic context. The current study applies this approach to evaluate the evolutionary status of the leaf beetle Calligrapha multipunctata variety suturella, a taxon described from a single locality in New Hampshire, but not further discussed in the literature. Here, we report on the collection of material that greatly extends the geographic range of this taxon and on the analysis of cytochrome oxidase I mitochondrial DNA sequences from these specimens and related Calligrapha species. We find that sequences from geographically overlapping populations of C. m. suturella and C. m. bigsbyana form two distinct, phylogenetically divergent clades that exhibit appreciable differentiation (4.5%) at the nucleotide level. This mtDNA differentiation was also evident in each of three localities ...

Since the description of Calligrapha scalaris (LeConte, 1824), a leaf beetle reported as an occasional serious pest of American elm, this taxon has been affected by remarkable taxonomic confusion. Most authors have invoked their particular and generally flawed concept of C. scalaris (LeConte) without reference to John E. LeConte's type material at the Museum of Comparative Zoology (Cambridge, Massachusetts). In this study, I have revised the series for C. scalaris (LeConte) conserved as part of J. LeConte and G. Horn collections, and confirmed that it includes as many as nine Calligrapha species, most of them lacking an association with Ulmus. Among these specimens, a couple of them from Texas are recognized as generally consistent with the original description and the only potentially elm-feeding animals. Thus, despite legitimate doubts for their availability to J. LeConte at the moment of the species description, they are designated here as neo- and paraneotypes, respectively, in an effort to maximize taxonomic stability. The species is redescribed on the basis of the neotype and diagnosed from all other species in this group, defined here as the “Calligrapha scalaris” group, including a provisional identification key for 14 species and for specimens conforming to the respective types. Finally, the study of syntype material from the Museum für naturkunde (Berlin) of C. multiguttis (Stål, 1865), an early synonym of C. scalaris (LeConte), allowed recognition of several other Calligrapha taxa affected by this synonymy, including C. ignota Brown, C. knabi Brown, and C. tiliae Brown ( = Chrysomela multiguttis Stål [pars] nov. syn.).