Current knowledge on the diversity of Eumolpinae (Coleoptera, Chrysomelidae) in New Caledonia.

The Eumolpinae leaf beetles of New Caledonia are very diverse, but our knowledge about their diversity is still incomplete. Following a renewed interest in the group in the last two decades, there has been an exponential increase in the number of species described, with species descriptions and taxonomic reassessment ongoing. In this work, the catalogue of New Caledonian Eumolpinae is updated, incorporating all these recent changes, and also indicating the collection where type specimens are currently available. The updated catalogue includes 120 species in 13 genera, and more additions and taxonomic changes, including new combinations, are expected in forthcoming years. Here two new synonymies are reported, namely Dumbeastriata Jolivet, Verma & Mille, 2007 = Taophilacancellata Samuelson, 2010, syn. nov.; and Dematochromatheryi Jolivet, Verma & Mille, 2010 = Dematochromapoyensis Jolivet, Verma & Mille, 2010, syn. nov. Moreover, two species still retaining their original adscription to the genus Colaspis Fabricius, 1801, are treated as incertae sedis. This catalogue represents a useful tool for future taxonomic studies of New Caledonian Chrysomelidae and can assist biodiversity surveys and conservation studies within the archipelago.

Analysis of intrinsic evolutionary factors leading to microendemic distributions in New Caledonian leaf beetles.

Microendemicity, or the condition of some species having local ranges, is a relatively common pattern in nature. However, the factors that lead to this pattern are still largely unknown. Most studies addressing this issue tend to focus on extrinsic factors associated with microendemic distributions, such as environmental conditions, hypothesising a posteriori about underlying potential speciation mechanisms, linked or not to these conditions. Here, we use a multi-faceted approach mostly focusing on intrinsic factors instead, namely diversification dynamics and speciation modes in two endemic sibling genera of leaf beetles with microendemic distributions, Taophila and Tricholapita, in a microendemicity hotspot, New Caledonia. Results suggest that the diversification rate in this lineage slowed down through most of the Neogene and consistently with a protracted speciation model possibly combined with several ecological and environmental factors potentially adding rate-slowing effects through time. In turn, species accumulated following successive allopatric speciation cycles, possibly powered by marked geological and climatic changes in the region in the last 25 million years, with daughter species ranges uncorrelated with the time of speciation. In this case, microendemicity seems to reflect a mature state for the system, rather than a temporary condition for recent species, as suggested for many microendemic organisms.

New Genera and Species Records of Nicaraguan Eumolpinae (Coleoptera: Chrysomelidae) Including a New Species in a New Generic Record for Central America.

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.

Systematic revision of Calligrapha s. str/. Chevrolat (Coleoptera: Chrysomelidae, Chrysomelinae).

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.).

Food Resource Sharing of Alder Leaf Beetle Specialists (Coleoptera: Chrysomelidae) as Potential Insect-Plant Interface for Horizontal Transmission of Endosymbionts.

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.

A new species of the genus Tricholapita nom. nov. and stat. nov. (Coleoptera: Chrysomelidae, Eumolpinae) from New Caledonia.

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.

Molecular phylogenetic assessment of the tribal classification of Lamiinae (Coleoptera: Cerambycidae).

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.

Patterns and Constraints in the Evolution of Sperm Individualization Genes in Insects, with an Emphasis on Beetles.

Gene expression profiles can change dramatically between sexes and sex bias may contribute specific macroevolutionary dynamics for sex-biased genes. However, these dynamics are poorly understood at large evolutionary scales due to the paucity of studies that have assessed orthology and functional homology for sex-biased genes and the pleiotropic effects possibly constraining their evolutionary potential. Here, we explore the correlation of sex-biased expression with macroevolutionary processes that are associated with sex-biased genes, including duplications and accelerated evolutionary rates. Specifically, we examined these traits in a group of 44 genes that orchestrate sperm individualization during spermatogenesis, with both unbiased and sex-biased expression. We studied these genes in the broad evolutionary framework of the Insecta, with a particular focus on beetles (order Coleoptera). We studied data mined from 119 insect genomes, including 6 beetle models, and from 19 additional beetle transcriptomes. For the subset of physically and/or genetically interacting proteins, we also analyzed how their network structure may condition the mode of gene evolution. The collection of genes was highly heterogeneous in duplication status, evolutionary rates, and rate stability, but there was statistical evidence for sex bias correlated with faster evolutionary rates, consistent with theoretical predictions. Faster rates were also correlated with clocklike (insect amino acids) and non-clocklike (beetle nucleotides) substitution patterns in these genes. Statistical associations (higher rates for central nodes) or lack thereof (centrality of duplicated genes) were in contrast to some current evolutionary hypotheses, highlighting the need for more research on these topics.

Assessment of the role of Wolbachia in mtDNA paraphyly and the evolution of unisexuality in Calligrapha (Coleoptera: Chrysomelidae).

Calligrapha is a New World leaf beetle genus that includes several unisexual species in northeastern North America. Each unisexual species had an independent hybrid origin involving different combinations of bisexual species. However, surprisingly, they all cluster in a single mtDNA clade and with some individuals of their parental species, which are in turn deeply polyphyletic for mtDNA. This pattern is suggestive of a selective sweep which, together with mtDNA taxonomic incongruence and occurrence of unisexuality in Calligrapha, led to hypothesize that Wolbachia might be responsible. I tested this hypothesis studying the correlation between diversity of Wolbachia and well-established mtDNA lineages in >500 specimens of two bisexual species of Calligrapha and their derived unisexual species. Wolbachia appears highly prevalent (83.4%), and fifteen new supergroup-A strains of the bacteria are characterized, belonging to three main classes: wCallA, occupying the whole species ranges, and wCallB and wCallC, narrowly parapatric, infecting beetles with highly divergent mtDNAs where they coexist. Most beetles (71.6%) carried double infections of wCallA with another sequence class. Bayesian inference of ancestral character states and association tests between bacterial diversity and the mtDNA genealogy show that each mtDNA lineage of Calligrapha has specific types of infection. Moreover, shifts can be explained by horizontal or vertical transfer from local populations to an expanding lineage and cytoplasmic incompatibility between wCallB and wCallC types, suggesting that the symbionts hitchhike with the host and are not responsible for selective mtDNA sweeps. Lack of evidence for sweeps and the fact that individuals in the unisexual clade are uninfected or infected by the widespread wCallA type indicate that Wolbachia does not induce unisexuality in Calligrapha, although they may manipulate host reproduction through cytoplasmic incompatibility.

Description of Kumatoeides gen. nov. (Coleoptera: Chrysomelidae, Eumolpinae) from New Caledonia.

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.

Systematic revision of Central American Calligrapha Chevrolat of the subgenus Erythrographa subgen. nov. (Coleoptera: Chrysomelidae, Chrysomelinae).

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.

Evaluation of bias on the assessment of diet breadth of herbivorous insects using molecular methods.

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.

Testis-specific RNA-Seq of Calligrapha (Chrysomelidae) as a transcriptomic resource for male-biased gene inquiry in Coleoptera.

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.

Insights on the genus Acronymolpus Samuelson with new synonymies and exclusion of Stethotes Baly from the fauna of New Caledonia (Coleoptera, Chrysomelidae, Eumolpinae).

In this work, several taxonomic problems affecting the recently erected genus Acronymolpus Samuelson, 2015, endemic to New Caledonia, are addressed. Two of the three New Caledonian species described in Stethotes Baly are transferred to Acronymolpus and their priority is recognized over the names proposed in the revision of this genus. Moreover, different forms of Acronymolpus always found in sympatry, one reddish and larger, and the other black and smaller, were each given species status in that revision, but they are recognized here as the females and males, respectively, of the same species. The taxonomic summary of these discoveries is: (i) A. bertiae (Jolivet, Verma & Mille, 2007), comb. n. = A. meteorus Samuelson, 2015, syn. n., and A. turbo Samuelson, 2015, syn. n.; and (ii) A. jourdani (Jolivet, Verma & Mille, 2013), comb. n. = A. gressitti Samuelson, 2015, syn. n., and A. joliveti Samuelson, 2015, syn. n. New distribution data and the male genitalia and the spermatheca of the two valid species of Acronymolpus are described for the first time with reference to taxonomically important characters. Finally, the last New Caledonian species described in Stethotes is recognized here as a member of the endemic genus Taophila Heller: T. mandjeliae (Jolivet, Verma & Mille, 2010), comb. n.

High-throughput biodiversity analysis: Rapid assessment of species richness and ecological interactions of Chrysomelidae (Coleoptera) in the tropics.

Biodiversity assessment has been the focus of intense debate and conceptual and methodological advances in recent years. The cultural, academic and aesthetic impulses to recognise and catalogue the diversity in our surroundings, in this case of living objects, is furthermore propelled by the urgency of understanding that we may be responsible for a dramatic reduction of biodiversity, comparable in magnitude to geological mass extinctions. One of the most important advances in this attempt to characterise biodiversity has been incorporating DNA-based characters and molecular taxonomy tools to achieve faster and more efficient species delimitation and identification, even in hyperdiverse tropical biomes. In this assay we advocate for a broad understanding of Biodiversity as the inventory of species in a given environment, but also the diversity of their interactions, with both aspects being attainable using molecular markers and phylogenetic approaches. We exemplify the suitability and utility of this framework for large-scale biodiversity assessment with the results of our ongoing projects trying to characterise the communities of leaf beetles and their host plants in several tropical setups. Moreover, we propose that approaches similar to ours, establishing the inventories of two ecologically inter-related and species-rich groups of organisms, such as insect herbivores and their angiosperm host-plants, can serve as the foundational stone to anchor a comprehensive assessment of diversity, also in tropical environments, by subsequent addition of trophic levels.

Systematic revision of Calligrapha Chevrolat (Coleoptera: Chrysomelidae) with pale spots on dark elytra and description of two new species.

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.

Subtle Ecological Gradient in the Tropics Triggers High Species-Turnover in a Local Geographical Scale.

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.

Systematic revision of the genus Calligrapha Chevrolat (Coleoptera: Chrysomelidae, Chrysomelinae) in Central America: The group of Calligrapha argus Stål.

Calligrapha Chevrolat is a large American leaf beetle genus of imprecise taxonomic boundaries. Most species occur in Central and South America where they have not been subject to modern systematic revisions. This work is the first attempt to clarify the systematics of the group by focusing on a relatively diverse species group well characterized from a morphological standpoint: the group of C. argus Stål. I provide a pragmatic identification key for major lineages in Calligrapha, highlighting a potentially useful character for supra-specific rank systematics within this genus, namely the shape of the distal end of the flagellum in the penis. This key helps recognizing the C. argus species group as one with species having a single spot enclosed by humeral lunule and a complete arcuate band, laterally confluent with subsutural stripe (except in C. elegantula Jacoby), deep hypomeral furrow and with characteristic ventral carving at apex of flagellum, as major distinguishing features. The species group is subdivided into fourteen species, whereby one previously described taxon, C. famularis Stål, is subordinated as subspecies of C. geographica Stål stat. nov., and two new species from southern Mexico and Guatemala are described: C. anabelae sp. nov. and C. catarinae sp. nov. An identification key for all the species in the group is provided, as well as collection data for the approximately 2,100 specimens determined for this work and distribution maps based on these collection data. The group is centred in the Caribbean Mesoamerican biogeographic domain, with two species reaching the California-Rocky Mountain domain to the north (C. ancoralis Stål and C. diversa Stål), and three species reaching the Caribbean Northwest-South American domain to the south (C. argus Stål, C. diversa Stål and C. simillima Stål).

Nonrandom patterns of genetic admixture expose the complex historical hybrid origin of unisexual leaf beetle species in the genus Calligrapha.

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.

Automated DNA-based plant identification for large-scale biodiversity assessment.

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.