Fungal endophytes can modulate plant invasion.

Symbiotic organisms may contribute to a host plant's success or failure to grow, its ability to maintain viable populations, and potentially, its probability of establishment and spread outside its native range. Intercellular and intracellular microbial symbionts that are asymptomatic in their plant host during some or all of their life cycle - endophytes - can form mutualistic, commensal, or pathogenic relationships, and sometimes novel associations with alien plants. Fungal endophytes are likely the most common endosymbiont infecting plants, with life-history, morphological, physiological, and plant-symbiotic traits that are distinct from other endophytic guilds. Here, we review the community dynamics of fungal endophytes during the process of plant invasion, and how their functional role may shift during the different stages of invasion: transport, introduction (colonisation), establishment, and spread. Each invasion stage presents distinct ecological filters that an alien plant must overcome to advance to the subsequent stage of invasion. Endophytes can alternately aid the host in overcoming stage-specific filters, or contribute to the barriers imposed by filters (e.g. biotic resistance), thereby affecting invasion pathways. A few fungi can be transported as seed endophytes from their native range and be vertically transmitted to future generations in the non-native range, especially in graminoids. In other plant groups, alien plants mostly acquire endophytes via horizontal transmission from the invaded plant community, and the host endophyte community is shaped by host filtering and biogeographic factors (e.g. dispersal limitation, environmental filtering). Endophytes infecting alien plants (both those transported with their host and those accumulated in the non-native range) may influence invasion success by affecting plant growth, reproduction, environmental tolerance, and pathogen and herbivory defences; however, the direction and magnitude of these effects can be contingent upon the host identity, life stage, ecological conditions, and invasion stage. This context dependence may cause endophytic fungi to shift to a non-endophytic (e.g. pathogenic) functional life stage in the same or different hosts, which can modify alien-native plant community dynamics. We conclude by identifying paths in which alien hosts can exploit the context dependency of endophyte function in novel abiotic and biotic conditions and at the different stages of invasion.

Phylogenomics reveals patterns of ancient hybridization and differential diversification that contribute to phylogenetic conflict in willows, poplars, and close relatives.

Despite the economic, ecological, and scientific importance of the genera Salix L. (willows) and Populus L. (poplars, cottonwoods, and aspens) Salicaceae, we know little about the sources of differences in species diversity between the genera and of the phylogenetic conflict that often confounds estimating phylogenetic trees. Salix subgenera and sections, in particular, have been difficult to classify, with one recent attempt termed a "spectacular failure" due to a speculated radiation of the subgenera Vetrix and Chamaetia. Here, we use targeted sequence capture to understand the evolutionary history of this portion of the Salicaceae plant family. Our phylogenetic hypothesis was based on 787 gene regions and identified extensive phylogenetic conflict among genes. Our analysis supported some previously described subgeneric relationships and confirmed the polyphyly of others. Using an fbranch analysis, we identified several cases of hybridization in deep branches of the phylogeny, which likely contributed to discordance among gene trees. In addition, we identified a rapid increase in diversification rate near the origination of the Vetrix-Chamaetia clade in Salix. This region of the tree coincided with several nodes that lacked strong statistical support, indicating a possible increase in incomplete lineage sorting due to rapid diversification. The extraordinary level of both recent and ancient hybridization in both Salix and Populus have played important roles in the diversification and diversity in these two genera.

The correct name for an Aquilegia (Ranunculaceae) hybrid of the parentage Aquilegiaflavescens × A.formosa.

Aquilegia×miniana (J.F.Macbr. & Payson) Cronk, hybr. & stat. nov. is the correct name for the hybrid Aquilegiaflavescens S.Watson × A.formosaFisch. & DC.var.formosa. In 1916 Payson and Macbride, while exploring the mountains of Idaho, found populations of Aquilegia that were pink in flower colour and appeared intermediate between the yellow-flowered A.flavescens and red-flowered A.formosa. They named these plants A.flavescensvar.miniana J.F.Macbr. & Payson. There has been uncertainty over whether their type collections (in GH, RM, MO, US, E, CM, CAS, NY) do indeed represent hybrids or pink-flowered morphs of A.flavescens. Using a Wells diagram, the holotype (in the Gray Herbarium of Harvard University) is shown to be intermediate, allowing its identification as a clear hybrid. However, some of the isotype material is indistinguishable from A.flavescens. The holotype matches material from British Columbia that has been determined to be of hybrid origin using molecular and morphological data. A.flavescensvar.miniana J.F.Macbr. & Payson is, therefore, an available name for the hybrid, which is here raised to the status of hybrid binomial.

Chloroplast markers for the Malvaceae and the plastome of Henderson's checkermallow (Sidalcea hendersonii S.Wats.), a rare plant from the Pacific Northwest.

OBJECTIVE: Sidalcea is a genus of flowering plants restricted to the west coast of North America, commonly known as checkermallows. Remarkably, of the ~ 30 recognized species, 16 are of conservation concern (vulnerable, imperilled or critically imperilled). To facilitate biological studies in this genus, and in the wider Malvaceae, we have sequenced the whole plastid genome of Sidalcea hendersonii. This will allow us both to check those regions already developed as general Malvaceae markers in a previous study, and to search for new regions. RESULTS: By comparing the Sidalcea genome to that of Althaea, we have identified a hypervariable circa 1 kb region in the short single copy region. This region shows promise for examining phylogeographic pattern, hybridization and haplotype diversity. Remarkably, considering the conservation of plastome architecture between Sidalcea and Althaea, the former has a 237 bp deletion in the otherwise highly conserved inverted repeat region. Newly designed primers provide a PCR assay to determine presence of this indel across the Malvaceae. Screening of previously designed chloroplast microsatellite markers indicates two markers with variation within S. hendersonii that would be useful in future population conservation genetics.

Report of two distinct ribotypes in ITS sequences of Phalarisarundinacea (Poaceae) in western Canada and Alaska.

Background: Phalarisarundinacea L. (reed canary grass) is a widely occurring grass throughout the Northern Hemisphere. In North America, it is thought to consist of introduced agricultural forms from Europe as well as native populations. New information: During a survey of Phalarisarundinacea in western Canada, we discovered two distinct ribotypes in the sequences of the internal transcribed spacer (ITS) of the nuclear ribosomal DNA: one full length (ITS-long) and one with a seven base pair deletion (ITS-short). In addition, ITS-long plants have fixed heterozygosity indicating possible polyploidy. Phylogenetic analysis reveals that ITS-short is a unique ribotype that characterises an intraspecific clade. We designed an efficient PCR-based assay that allows sizing of a 238/245 base pair fragment in a capillary sequencer. This approach provides a novel marker that could be useful in future surveys of Phalarisarundinacea.

Floral phenology of an Andean bellflower and pollination by buff-tailed sicklebill hummingbird.

The Andean bellflowers comprise an explosive radiation correlated with shifts to specialized pollination. One diverse clade has evolved with extremely curved floral tubes and is predicted to be pollinated exclusively by one of two parapatric species of sicklebill hummingbirds (Eutoxeres). In this study, we focused on the floral biology of Centropogon granulosus, a bellflower thought to be specialized for pollination by Eutoxeres condamini, in a montane cloud forest site in southeastern Peru. Using camera traps and a pollination exclusion experiment, we documented E. condamini as the sole pollinator of C. granulosus. Visitation by E. condamini was necessary for fruit development. Flowering rates were unequivocally linear and conformed to the "steady-state" phenological type. Over the course of >1800 h of monitoring, we recorded 12 E. condamini visits totaling 42 s, indicating traplining behavior. As predicted by its curved flowers, C. granulosus is exclusively pollinated by buff-tailed sicklebill within our study area. We present evidence for the congruence of phenology and visitation as a driver of specialization in this highly diverse clade of Andean bellflowers.

Some sexual consequences of being a plant.

Plants have characteristic features that affect the expression of sexual function, notably the existence of a haploid organism in the life cycle, and in their development, which is modular, iterative and environmentally reactive. For instance, primary selection (the first filtering of the products of meiosis) is via gametes in diplontic animals, but via gametophyte organisms in plants. Intragametophytic selfing produces double haploid sporophytes which is in effect a form of clonal reproduction mediated by sexual mechanisms. In homosporous plants, the diploid sporophyte is sexless, sex being only expressed in the haploid gametophyte. However, in seed plants, the timing and location of gamete production is determined by the sporophyte, which therefore has a sexual role, and in dioecious plants has genetic sex, while the seed plant gametophyte has lost genetic sex. This evolutionary transition is one that E.J.H. Corner called 'the transference of sexuality'. The iterative development characteristic of plants can lead to a wide variety of patterns in the distribution of sexual function, and in dioecious plants poor canalization of reproductive development can lead to intrasexual mating and the production of YY supermales or WW superfemales. Finally, plant modes of asexual reproduction (agamospermy/apogamy) are also distinctive by subverting gametophytic processes. This article is part of the theme issue 'Sex determination and sex chromosome evolution in land plants'.

The distribution of sexual function in the flowering plant: from monoecy to dioecy.

In flowering plants, male and female functions are usually closely associated in the same flowers, as predicted by resource allocation theory. However, the benefits of outbreeding can lead to unisexual flowers and the physiological control of their distribution across the plant (monoecy). Monoecy is thought to be a major route to dioecy (separation of sexual function of different individuals). The developmental and functional problems associated with unisexual flowers may thus be solved at the level of the evolution of monoecy. Consequently, the evolution of dioecy from monoecy requires mutations in only a single gene. Here various scenarios (conceptual models) are presented for the evolution of monoecy and dioecy, including scenarios consistent with known cases of single-gene control of dioecy, such as in Populus, and the artificial breeding of dioecy from monoecy experimentally achieved in Zea and Cucumis. Attention is also drawn here to the phenomenon of pleogamy, the minor or occasional occurrence of additional sex morphs within a species, which may provide important information about the genetic and developmental control of various sexual systems. This article is part of the theme issue 'Sex determination and sex chromosome evolution in land plants'.

Plant-Pollinator Specialization: Origin and Measurement of Curvature.

AbstractA feature of biodiversity is the abundance of curves displayed by organs and organisms. Curvature is a widespread, convergent trait that has important ecological and evolutionary implications. In pollination ecology, the curvature of flowers and pollinator mouthparts (e.g., hummingbird bills) along the dorsiventral plane has been associated with specialization, competition, and species coexistence. Six differing methods have historically been used to measure curvature in pollination systems; we provide a solution to this inconsistency by defining curvature using well-established concepts from differential geometry. Intuitively, curvature is the degree to which a line is not straight, but more formally it is the rate at which the tangent of a curve changes direction with respect to arc length. Here, we establish a protocol wherein a line is fitted against landmarks placed on an image of a curved organ or organism, then curvature is computed at many points along the fitted line and the sum taken. The protocol is demonstrated by studying the development of nectar spur curvature in the flowering plant genus Epimedium (Berberidaceae). By clarifying the definition of curvature, our aim is to make the language of comparative morphology more precise and broadly applicable to capture other curved structures in nature.

Whole-genome sequencing and genome regions of special interest: Lessons from major histocompatibility complex, sex determination, and plant self-incompatibility.

Whole-genome sequencing of non-model organisms is now widely accessible and has allowed a range of questions in the field of molecular ecology to be investigated with greater power. However, some genomic regions that are of high biological interest remain problematic for assembly and data-handling. Three such regions are the major histocompatibility complex (MHC), sex-determining regions (SDRs) and the plant self-incompatibility locus (S-locus). Using these as examples, we illustrate the challenges of both assembling and resequencing these highly polymorphic regions and how bioinformatic and technological developments are enabling new approaches to their study. Mapping short-read sequences against multiple alternative references improves genotyping comprehensiveness at the S-locus thereby contributing to more accurate assessments of allelic frequencies. Long-read sequencing, producing reads of several tens to hundreds of kilobase pairs in length, facilitates the assembly of such regions as single sequences can span the multiple duplicated gene copies of the MHC region, and sequence through repetitive stretches and translocations in SDRs and S-locus haplotypes. These advances are adding value to short-read genome resequencing approaches by allowing, for example, more accurate haplotype phasing across longer regions. Finally, we assessed further technical improvements, such as nanopore adaptive sequencing and bioinformatic tools using pangenomes, which have the potential to further expand our knowledge of a number of genomic regions that remain challenging to study with classical resequencing approaches.

Dark extinction: the problem of unknown historical extinctions.

The extinction of species before they are discovered and named (dark extinction, DE) is widely inferred as a significant part of species loss in the 'pre-taxonomic' period (approx. 1500-1800 CE) and, to some extent, in the 'taxonomic period' (approx. 1800-present) as well. The discovery of oceanic islands and other pristine habitats by European navigators and the consequent introduction of destructive mammals, such as rats and goats, started a process of anthropogenic extinction. Much ecosystem change happened before systematic scientific recording, so has led to DE. Statistical methods are available to robustly estimate DE in the 'taxonomic period'. For the 'pre-taxonomic period', simple extrapolation can be used. The application of these techniques to world birds, for example, suggests that approximately 56 DEs occurred in the 'taxonomic period' (1800-present) and approximately 180 in the 'pre-taxonomic period' (1500-1800). Targeting collection activities in extinction hotspots, to make sure organisms are represented in collections before their extinction, is one way of reducing the number of extinct species without a physical record (providing that collection efforts do not themselves contribute to species extinction).

A targeted sequence capture array for phylogenetics and population genomics in the Salicaceae.

PREMISE: The family Salicaceae has proved taxonomically challenging, especially in the genus Salix, which is speciose and features frequent hybridization and polyploidy. Past efforts to reconstruct the phylogeny with molecular barcodes have failed to resolve the species relationships of many sections of the genus. METHODS: We used the wealth of sequence data in the family to design sequence capture probes to target regions of 300-1200 bp of exonic regions of 972 genes. RESULTS: We recovered sequence data for nearly all of the targeted genes in three species of Populus and three species of Salix. We present a species tree, discuss concordance among gene trees, and present population genomic summary statistics for these loci. CONCLUSIONS: Our sequence capture array has extremely high capture efficiency within the genera Populus and Salix, resulting in abundant phylogenetic information. Additionally, these loci show promise for population genomic studies.

Default Sex and Single Gene Sex Determination in Dioecious Plants.

A well-established hypothesis for the evolution of dioecy involves two genes linked at a sex-determining region (SDR). Recently there has been increased interest in possible single gene sex determination. Work in Populus has finally provided direct experimental evidence for single gene sex determination in plants using CRISPR-Cas9 to knock out a single gene and convert individuals from female to male. In poplar, the feminizing factor popARR17 acts as a "master regulator", analogous to the mammalian masculinizing factor SRY. The production of fully functional males from females by a simple single gene knockout is experimental evidence that an antagonistic male-determining factor does not exist in Populus. Mammals have a "default sex" (female), as do poplar trees (Populus), although the default sex in poplars is male. The occurrence of single gene sex determination with a default sex may be much commoner in plants than hitherto expected, especially when dioecy evolved via monoecy. The master regulator does not even need to be at the SDR (although it may be). In most poplars the feminizing factor popARR17 is not at the SDR, but instead a negative regulator of it. So far there is little information on how high-level regulators are connected to floral phenotype. A model is presented of how sex-determining genes could lead to different floral morphologies via MADS-box floral developmental genes.

Functional Characterization of Terpene Synthases Accounting for the Volatilized-Terpene Heterogeneity in Lathyrus odoratus Cultivar Flowers.

Lathyrus odoratus (sweet pea) is an ornamental plant with exceptional floral scent, previously used as an experimental organism in the early development of Mendelian genetics. However, its terpene synthases (TPSs), which act as metabolic gatekeepers in the biosynthesis of volatile terpenoids, remain to be characterized. Auto-Headspace Solid-phase Microextraction/Gas chromatography-mass spectrometry analysis of floral volatile terpene constituents from seven sweet pea cultivars identified α-bergamotene, linalool, (-)-α-cubebene, geraniol, ß-caryophyllene and ß-sesquiphellandrene as the dominant compounds. RNA sequencing was performed to profile the transcriptome of L. odoratus flowers. Bioinformatic analysis identified eight TPS genes (acronymed as LoTPS) that were successfully cloned, heterologously expressed and functionally analyzed. LoTPS4 and LoTPS7, belonging to the TPS-b clade, biochemically catalyzed the formation of monoterpenes and sesquiterpenes. LoTPS3 and LoTPS8, placed in the TPS-a clade, also generated monoterpenes and sesquiterpenes, while LoTPS12 belonging to the TPS-g clade showed linalool/nerolidol synthase activity. Notably, biochemical assays of the recombinant LoTPS proteins revealed their catalytic promiscuity, and the enzymatic products were basically consistent with major volatile compounds released from sweet pea flowers. The data from our study lay the foundation for the chemical ecology, molecular genetics and biotechnological improvement of sweet pea and other legumes (Fabaceae).

Salix transect of Europe: patterns in the distribution of willow-feeding psyllids (Hemiptera: Psylloidea) from Greece to arctic Norway.

Background Psyllids are oligophagous phytophagous insects with many specialist willow (Salix spp.) feeding species in two genera (Cacopsylla and Bactericera). We examine the patterns of distribution and co-occurrence of willow-feeding species at 42 willow sites across Europe forming a transect from Greece (lat. 38.8 °N) to arctic Norway (lat. 70.6 °N). The transect and sites have been described in previous papers. New information A total of 1245 individual psyllids were examined from 23 species of willow over the transect, representing 17 willow-feeding species (11 Cacopsylla and 6 Bactericera). Numerous species were very widely distributed, with two species, Bactericera albiventris (Foerster, 1848) and Cacopsylla pulchra (Zetterstedt, 1840), occurring from Greece to Finland. Other widespread species (Romania to Finland) were Cacopsylla ambigua (Foerster, 1848) and Bactericera curvatinervis (Foerster, 1848). The mean number of psyllid species per site was 2.4 (1.3 Cacopsylla, 1.1 Bactericera).

Salix transect of Europe: records of willow-associated weevils (Coleoptera: Curculionoidea) from Greece to Arctic Norway, with insights from DNA barcoding.

BACKGROUND: Curculionid beetles associated with willow (Salix spp.) were surveyed at 42 sites across Europe, from Greece (lat. 38.8 °N) to arctic Norway (lat. 69.7 °N). DNA sequence data provide additional verification of identifications and geographic clustering. NEW INFORMATION: In all, 73 curculionid species were collected from willows, of which seven were particularly abundant. The most widespread species were: Acalyptus carpini Fabricius, 1793 at 15 sites; Tachyerges stigma Germar, 1821 at 13 sites; Phyllobius oblongus (Linnaeus, 1758) at 11 sites; Phyllobius maculicornis Germar, 1824 at 10 sites; and Archarius salicivorus (Paykull, 1792), Melanapion minimum (Herbst, 1797), and Phyllobius cf. pyri (Linnaeus, 1758) all at nine sites. The mean number of curculionid species collected on willow at each site was 5.5 (range 0-14). Compared to chrysomelids, curculionids were richer in species but the species had relatively low average abundance. Widespread curculionid species appear to have scattered and patchy observed distributions with limited geographical structuring in our data. However, deeper sampling (e.g. over multiple seasons and years), would give a better indication of distribution, and may increase apparent geographical structuring. There is some site-to-site variation in colour in a few taxa, but little notable size variation. DNA barcoding, performed on some of the more common species, provides clear species clusters and definitive separation of the taxonomically more challenging species, as well as some interesting geographic insights. Our northernmost sample of Phyllobius oblongus is unique in clustering with Canadian samples of this species. On the other hand, our samples of Acalyptus carpini cluster with European samples and are distinct from a separate Canadian cluster of this species. We provide the first available DNA sequences for Phyllobius thalassinus Gyllenhal, 1834 (Hungary).

A single gene underlies the dynamic evolution of poplar sex determination.

Although hundreds of plant lineages have independently evolved dioecy (that is, separation of the sexes), the underlying genetic basis remains largely elusive1. Here we show that diverse poplar species carry partial duplicates of the ARABIDOPSIS RESPONSE REGULATOR 17 (ARR17) orthologue in the male-specific region of the Y chromosome. These duplicates give rise to small RNAs apparently causing male-specific DNA methylation and silencing of the ARR17 gene. CRISPR-Cas9-induced mutations demonstrate that ARR17 functions as a sex switch, triggering female development when on and male development when off. Despite repeated turnover events, including a transition from the XY system to a ZW system, the sex-specific regulation of ARR17 is conserved across the poplar genus and probably beyond. Our data reveal how a single-gene-based mechanism of dioecy can enable highly dynamic sex-linked regions and contribute to maintaining recombination and integrity of sex chromosomes.

Moth versus fly: a preliminary study of the pollination mode of two species of endemic Asteraceae from St Helena (Commidendrum robustum and C. rugosum) and its conservation implications.

Commidendrum robustum (Roxb.) DC. (St Helena gumwood) and C. rugosum (Dryand.) DC. (St Helena scrubwood) are ecologically important, endemic woody Asteraceae from the isolated South Atlantic island of St Helena. Once very abundant, they now exist in sparse fragmented populations due to 500 years of environmental destruction. They are sister taxa that evolved on the island and are reported to hybridise. Commidendrum rugosum has a saucer-like erect capitulum, whereas C. robustum has a somewhat globular hanging capitulum. Using daytime timelapse photography to follow capitula through their life cycle, we found that C. rugosum appears to be myophilous, visited largely by flies (including the endemic syrphid, Sphaerophoria beattiei Doesburg & Doesburg) and occasionally by Lepidoptera. Commidendrum robustum, on the other hand, although visited by flies, strongly attracts moths (especially noted at the Millennium Forest site). Our data suggest that moth visits may reduce visits from flies due to the sensitivity of flies to interference by other insects. We conclude that C. robustum may have a mixed syndrome of myophily/phalaenophily and that there is apparently some divergence of the pollination niche between the two species. Its potential in attracting moths, coupled with its former abundance, suggests that it may have been a major food source for adults of the numerous endemic moths. Pollinator activity was measured by insect visitation rates (mean visits per capitulum per day, V) and insect residence time (mean pollinator kiloseconds per capitulum per day, R). Both are higher for C. robustum (C. rugosum, V = 16.4, R = 3.101; C. robustum, V = 34.0, R = 8.274), reflecting the abundance of moths on the capitula at the Millennium Forest site. The conservation implications of the pollination mode are that: (1) there is considerable pollinator activity on the capitula and pollination is not currently a limiting factor for plant reproduction; (2) gene exchange between geographically-isolated populations of C. rugosum is likely to be minimal due to the apparent reliance of the species for pollination on small flies (especially Sphaerophoria beattiei), which are believed to be not effective as pollinators over long distances (> 1 km). A possible exception is the strong-flying drone-fly, Eristalis tenax Linn. which, although not as abundant as Sphaerophoria, does visit the flowers; (3) there is considerable overlap between the two species in flower visitors and interspecific pollen transfer is possible where the two species grow intermixed (which has potential positive and negative implications for species survival).

Gene expression trajectories during male and female reproductive development in balsam poplar (Populus balsamifera L.).

Plant reproductive development from the first appearance of reproductively committed axes through to floral maturation requires massive and rapid remarshalling of gene expression. In dioecious species such as poplar this is further complicated by divergent male and female developmental programs. We used seven time points in male and female balsam poplar (Populus balsamifera L.) buds and catkins representing the full annual flowering cycle, to elucidate the effects of time and sex on gene expression during reproductive development. Time (developmental stage) is dominant in patterning gene expression with the effect of sex nested within this. Here, we find (1) evidence for five successive waves of alterations to the chromatin landscape which may be important in setting the overall reproductive trajectory, regardless of sex. (2) Each individual developmental stage is further characterized by marked sex-differential gene expression. (3) Consistent sexually differentiated gene expression regardless of developmental stage reveal candidates for high-level regulators of sex and include the female-specific poplar ARR17 homologue. There is also consistent male-biased expression of the MADS-box genes PISTILLATA and APETALA3. Our work provides insights into expression trajectories shaping reproductive development, its potential underlying mechanisms, and sex-specific translation of the genome information into reproductive structures in balsam poplar.

Salix transect of Europe: additional leaf beetle (Chrysomelidae) records and insights from chrysomelid DNA barcoding.

Occurrence patterns of chrysomelid beetles (Coleoptera: Chrysomelidae), associated with willow (Salix spp.) at 42 sites across Europe, have previously been described. The sites form a transect from Greece (lat. 38.8 °N) to arctic Norway (lat. 69.7 °N). This paper reports additional records and the results of DNA sequencing in certain genera. Examination of further collections from the transect has added 13 species in the genera Aphthona, Chrysomela, Cryptocephalus, Epitrix, Galerucella (2 spp.), Gonioctena, Phyllotreta (2 spp.), Pachybrachis (3 spp.) and Syneta. We also report the sequencing of the DNA regions cytochrome oxidase 1 (CO1) and cytochrome B (cytB) for a number of samples in the genera Plagiodera, Chrysomela, Gonioctena, Phratora, Galerucella and Crepidodera. The cytB sequences are the first available for some of these taxa. The DNA barcoding largely confirmed previous identifications but allowed a small number of re-assignments between related species. Most notably, however, it was evident that the southernmost material (Greece and Bulgaria) of specimens, previously treated as Crepidodera aurata sens. lat., belonged to a distinctive molecular cluster. Morphological re-examination revealed these to be C. nigricoxis Allard, 1878. This is an example of how morphotaxonomy and DNA barcoding can work iteratively to refine identification. Our sequences for C. nigricoxis appear to be the first available for this taxon. Finally, there is little geographic structure evident, even in widely dispersed species.