Fish Parasite Dinoflagellates Haidadinium ichthyophilum and Piscinoodinium Share a Recent Common Ancestor

The dinoflagellate Haidadinium ichthyophilum Buckland‐Nicks, Reimchen and Garbary 1997 is an ectoparasite of the spine‐deficient, three‐spine stickleback Gasterosteus aculeatus L. Reimchen 1984, a fish endemic to Rouge Lake, Haida Gwaii. Haidadinium ichthyophilum proved difficult to assign taxonomically because its morphology and complex life cycle exhibited defining characteristics of both autotrophic and heterotrophic dinoflagellates, and was tentatively assigned to the Phytodiniales. Here, we characterized a 492 bp fragment of the small subunit ribosomal RNA (SSU rRNA) from preserved H. ichthyophilum cysts. In SSU phylogeny, H. ichthyophilum branches with the fish parasites, Piscinoodinium sp., strongly supporting the inclusion of H. ichthyophilum within the Suessiales.     

A polyphasic approach to the delimitation of diatom species: a case study for the genus Pinnularia (Bacillariophyta)

Diatoms are one of the most abundant and arguably the most species‐rich group of protists. Diatom species delimitation has often been based exclusively on the recognition of morphological discontinuities without investigation of other lines of evidence. Even though DNA sequences and reproductive experiments have revealed several examples of (pseudo)cryptic diversity, our understanding of diatom species boundaries and diversity remains limited. The cosmopolitan pennate raphid diatom genus Pinnularia represents one of the most taxon‐rich diatom genera. In this study, we focused on the delimitation of species in one of the major clades of the genus, the Pinnularia subgibba group, based on 105 strains from a worldwide origin. We compared genetic distances between the sequences of seven molecular markers and selected the most variable pair, the mitochondrial cox1 and nuclear encoded LSU rDNA, to formulate a primary hypothesis on the species limits using three single‐locus automated species delimitation methods. We compared the DNA‐based primary hypotheses with morphology and with other available lines of evidence. The results indicate that our data set comprised 15 species of the P. subgibba group. The vast majority of these taxa have an uncertain taxonomic identity, suggesting that several may be unknown to science and/or members of (pseudo)cryptic species complexes within the P. subgibba group.     

Phylogeny of Gracilariaceae (Rhodophyta): evidence from plastid and mitochondrial nucleotide sequences

Gracilariaceae are mostly pantropical red algae and include ~230 species in seven genera. Infrafamilial classification of the group has long been based on reproductive characters, but previous phylogenies have shown that traditionally circumscribed groups are not monophyletic. We performed phylogenetic analyses using two plastid (universal plastid amplicon and rbcL) and one mitochondrial (cox1) loci from a greatly expanded number of taxa to better assess generic relationships and understand patterns of character distributions. Our analyses produce the most well‐supported phylogeny of the family to date, and indicate that key characteristics of spermatangia and cystocarp type do not delineate genera as commonly suggested. Our results further indicate that Hydropuntia is not monophyletic. Given their morphological overlap with closely related members of Gracilaria, we propose that Hydropuntia be synonymized with the former. Our results additionally expand the known ranges of several Gracilariaceae species to include Brazil. Lastly, we demonstrate that the recently described Gracilaria yoneshigueana should be synonymized as G. domingensis based on morphological and molecular characters. These results demonstrate the utility of DNA barcoding for understanding poorly known and fragmentary materials of cryptic red algae.     

Biodiversity and Molecular Phylogeny of Australian Clevelandella Species (Class Armophorea,Order Clevelandellida,Family Clevelandellidae), Intestinal Endosymbiotic Ciliates in the Wood‐Feeding Roach Panesthia cribrata Saussure, 1864

There are over 100 species in the Order Clevelandellida distributed in many hosts. The majority is assigned to one of the five families, the Nyctotheridae. Our knowledge of clevelandellid genetic diversity is limited to species of Nyctotherus and Nyctotheroides. To increase our understanding of clevelandellid genetic diversity, species were isolated from intestines of the Australian wood‐feeding roach Panesthia cribrata Saussure, 1864 from August to October, 2008. Four morphospecies, similar to those reported in Java and Japan by Kidder [Parasitologica, 29 :163–205], were identified: Clevelandella constricta, Clevelandella nipponensis, Clevelandella parapanesthiae, and Clevelandella panesthiae. Small subunit rRNA gene sequences assigned all species to a “family” clade that was sister to the clade of species assigned to the Family Nyctotheridae in the Order Clevelandellida. Genetics and morphology were consistent for the first three Clevelandella species, but isolates assigned to C. panesthiae were assignable to three different genotypes, suggesting that this may be a cryptic species complex.     

Ulva (Chlorophyta,Ulvales) Biodiversity in the North Adriatic Sea (Mediterranean,Italy): Cryptic Species and New Introductions

Ulva Linnaeus (Ulvophyceae, Ulvales) is a genus of green algae widespread in different aquatic environments. Members of this genus show a very simple morphology and a certain degree of phenotypic plasticity, heavily influenced by environmental conditions, making difficult the delineation of species by morphological features alone. Most studies dealing with Ulva biodiversity in Mediterranean waters have been based only on morphological characters and a modern taxonomic revision of this genus in the Mediterranean is not available. We report here the results of an investigation on the diversity of Ulva in the North Adriatic Sea based on molecular analyses. Collections from three areas, two of which subject to intense shipping traffic, were examined, as well as historical collections of Ulva stored in the Herbarium Patavinum of the University of Padova, Italy. Molecular analyses based on partial sequences of the rbcL and tufA genes revealed the presence of six different species, often with overlapping morphologies: U. californica Wille, U. flexuosa Wulfen, U. rigida C. Agardh, U. compressa Linnaeus, U. pertusa Kjellman, and one probable new taxon. U. californica is a new record for the Mediterranean and U. pertusa is a new record for the Adriatic. Partial sequences obtained from historical collections show that most of the old specimens are referable to U. rigida. No specimens referable to the two alien species were found among the old herbarium specimens. The results indicate that the number of introduced seaweed species and their impact on Mediterranean communities have been underestimated, due to the difficulties in species identification of morphologically simple taxa as Ulva.     

Common genetic variants in GAL,GAP43 and NRSN1 and interaction networks confer susceptibility to Hirschsprung disease

Hirschsprung disease (HSCR) is a severe multifactorial genetic disorder. Microarray studies indicated GAL, GAP43 and NRSN1 might contribute to the altered risk in HSCR. Thus, we focused on genetic variations in GAL, GAP43 and NRSN1, and the gene‐gene interactions involved in HSCR susceptibility. We recruited a strategy combining case‐control study and MassArray system with interaction network analysis. For GAL, GAP43 and NRSN1, a total of 18 polymorphisms were assessed in 104 subjects with sporadic HSCR and 151 controls of Han Chinese origin. We found statistically significant differences between HSCR and control groups at 5 genetic variants. For each gene, the haplotypes combining all polymorphisms were the most significant. Based on SNPsyn, MDR and GeneMANIA analyses, we observed significant gene‐gene interactions among GAL, GAP43, NRSN1 and our previous identified RELN, GABRG2 and PTCH1. Our study for the first time indicates that genetic variants within GAL, GAP43 and NRSN1 and related gene‐gene interaction networks might be involved in the altered susceptibility to HSCR in the Han Chinese population, which might shed more light on HSCR pathogenesis.     

Phylogeography and diversification of an Amazonian understorey hummingbird: paraphyly and evidence for widespread cryptic speciation in the Plio‐Pleistocene

Straight‐billed Hermit Phaethornis bourcieri inhabits the understorey of upland terra firme forest throughout most of the Amazon basin. Currently, two allopatric taxa regarded as subspecies are recognized: P. b. bourcieri and P. b. major. However, the validity, interspecific limits and evolutionary history of these taxa are not yet fully elucidated. We use molecular characters to propose a phylogenetic hypothesis for populations and taxa grouped under Phaethornis bourcieri. Our results showed that P. bourcieri is part of the ‘Ametrornis’ clade, along with Phaethornis philippii and Phaethornis koepckeae, and that the subspecies major is more closely related to the latter two species than to populations grouped under nominate bourcieri. Our phylogenetic hypotheses recovered three main reciprocally monophyletic clades under nominate bourcieri separated by the lower Negro River and the Branco River or the Branco–Negro interfluve (clades B and C) and the upper Amazon (Solimões) or lower Marañon/Ucayali Rivers (clades C and D). Based on multi‐locus phylogeographic and population genetics approaches, we show that P. b. major is best treated as a separate species, and that P. b. bourcieri probably includes more than one evolutionary species, whose limits remain uncertain. The diversification of the ‘Ametrornis’ clade (P. bourcieri, P. philippii and P. koepckeae) is centred in the Amazon and appears to be closely linked to the formation of the modern Amazon drainage during the Plio‐Pleistocene.     

Hidden diversity and cryptic speciation refute cosmopolitan distribution in Caprella penantis (Crustacea: Amphipoda: Caprellidae)

Caprella penantis is considered a cosmopolitan species and one of the most challenging caprellids in taxonomic terms because of its remarkable intraspecific morphological variation. This study examined DNA sequences from mitochondrial (COI) and nuclear (18S) markers together with morphological data from 25 localities of C. penantis, and closely related species Caprella dilatata and Caprella andreae, all traditionally considered part of the old ‘acutifrons’ complex. The large genetic divergence and reciprocally allopatric distributions point to the existence of a species complex of at least four species, of which one is reported as a cryptic species. This study provides the first evidence of cryptic speciation in the family Caprellidae, and questions the validity of some traditional morphological characters used to delimit species in the genus Caprella. Our results are consistent with the idea that main factors were probably isolation by distance and ecological traits, promoting diversification in C. penantis. The strong genetic structure reported for this species in the Iberian Peninsula and Moroccan coasts also suggests restriction to dispersal as well as the presence of refugial areas. These results highlight the utility of the COI and 18S genes in combination with morphological characters for shedding light on systematic questions in caprellids, and patterns of genetic connectivity.     

Barley whole exome capture: a tool for genomic research in the genus Hordeum and beyond

Advanced resources for genome‐assisted research in barley (Hordeum vulgare) including a whole‐genome shotgun assembly and an integrated physical map have recently become available. These have made possible studies that aim to assess genetic diversity or to isolate single genes by whole‐genome resequencing and in silico variant detection. However such an approach remains expensive given the 5 Gb size of the barley genome. Targeted sequencing of the mRNA‐coding exome reduces barley genomic complexity more than 50‐fold, thus dramatically reducing this heavy sequencing and analysis load. We have developed and employed an in‐solution hybridization‐based sequence capture platform to selectively enrich for a 61.6 megabase coding sequence target that includes predicted genes from the genome assembly of the cultivar Morex as well as publicly available full‐length cDNAs and de novo assembled RNA‐Seq consensus sequence contigs. The platform provides a highly specific capture with substantial and reproducible enrichment of targeted exons, both for cultivated barley and related species. We show that this exome capture platform provides a clear path towards a broader and deeper understanding of the natural variation residing in the mRNA‐coding part of the barley genome and will thus constitute a valuable resource for applications such as mapping‐by‐sequencing and genetic diversity analyzes.     

Interactions Between Rhizoctonia Root Rot and the Foliar Common Bean Diseases Anthracnose and Rust

The effects of co‐inoculation of Rhizoctonia solani and Colletotrichum lindemuthianum or Uromyces appendiculatus at different inoculum levels were studied on the disease dynamics and on the growth of bean plants under greenhouse conditions. Bean seeds were sown in R. solani‐infested soil. Additional experiments in which seedlings were transplanted to infested soil were also carried out. Conidial suspensions of C. lindemuthianum or uredospores of U. appendiculatus were inoculated onto leaves at plant developmental stages V2 and V3, respectively. Interactions between root rot and the aerial diseases were observed depending on the inoculum levels and on the timing of R. solani inoculation. Anthracnose severity tended to be higher on R. solani‐infected plants. Conversely, R. solani infection significantly reduced diameter of pustules and rust severity. When seedlings were transplanted to soil infested with low levels of R. solani, root rot severity and density of R. solani in the soil were magnified at high levels of C. lindemuthianum or U. appendiculatus. In these experiments, a synergistic interaction between root rot and anthracnose was observed to affect the plant dry weight. Antagonistic effects on the plant dry weight were found for the combination root rot/rust only when seeds were sown in infested soil.     

Functional screening of willow alleles in Arabidopsis combined with QTL mapping in willow (Salix) identifies SxMAX4 as a coppicing response gene

Willows (Salix spp.) are important biomass crops due to their ability to grow rapidly with low fertilizer inputs and ease of cultivation in short‐rotation coppice cycles. They are relatively undomesticated and highly diverse, but functional testing to identify useful allelic variation is time‐consuming in trees and transformation is not yet possible in willow. Arabidopsis is heralded as a model plant from which knowledge can be transferred to advance the improvement of less tractable species. Here, knowledge and methodologies from Arabidopsis were successfully used to identify a gene influencing stem number in coppiced willows, a complex trait of key biological and industrial relevance. The strigolactone‐related More AXillary growth (MAX) genes were considered candidates due to their role in shoot branching. We previously demonstrated that willow and Arabidopsis show similar response to strigolactone and that transformation rescue of Arabidopsis max mutants with willow genes could be used to detect allelic differences. Here, this approach was used to screen 45 SxMAX1, SxMAX2, SxMAX3 and SxMAX4 alleles cloned from 15 parents of 11 mapping populations varying in shoot‐branching traits. Single‐nucleotide polymorphism (SNP) frequencies were locus dependent, ranging from 29.2 to 74.3 polymorphic sites per kb. SxMAX alleles were 98%–99% conserved at the amino acid level, but different protein products varying in their ability to rescue Arabidopsis max mutants were identified. One poor rescuing allele, SxMAX4D, segregated in a willow mapping population where its presence was associated with increased shoot resprouting after coppicing and colocated with a QTL for this trait.     

Multi‐locus phylogeny of African pipits and longclaws (Aves: Motacillidae) highlights taxonomic inconsistencies

The globally distributed avian family Motacillidae consists of five to seven genera (Anthus, Dendronanthus, Tmetothylacus, Macronyx and Motacilla, and depending on the taxonomy followed, Amaurocichla and Madanga) and 66–68 recognized species, of which 32 species in four genera occur in sub‐Saharan Africa. The taxonomy of the Motacillidae has been contentious, with variable numbers of genera, species and subspecies proposed and some studies suggesting greater taxonomic diversity than currently recognized (five genera and 67 species). Using one nuclear (Mb) and two mitochondrial (cyt b and CO1) gene regions amplified from DNA extracted from contemporary and museum specimens, we investigated the taxonomic status of 56 of the currently recognized motacillid species and present the most taxonomically complete and expanded phylogeny of this family to date. Our results suggest that the family comprises six clades broadly reflecting continental distributions: sub‐Saharan Africa (two clades), the New World (one clade), Palaearctic (one clade), a widespread large‐bodied Anthus clade, and a sixth widespread genus, Motacilla. Within the Afrotropical region, our phylogeny further supports recognition of Wood Pipit Anthus nyassae as a valid species, and the treatment of Long‐tailed Pipit Anthus longicaudatus and Kimberley Pipit Anthus pseudosimilis as junior subjective synonyms of Buffy Pipit Anthus vaalensis and African Pipit Anthus cinnamomeus, respectively. As the disjunct populations of Long‐billed Pipit Anthus similis in southern and East Africa are genetically distinct and geographically separated, we propose a specific status for the southern African population under the earliest available name, Nicholson's Pipit Anthus nicholsoni. Further, as our analyses indicate that Yellow‐breasted Pipit Anthus chloris and Golden Pipit Tmetothylacus tenellus are both nested within the Macronyx longclaws, we propose transferring these species to the latter genus.     

Absence from the nest due to human disturbance induces higher nest predation risk than natural recesses in Common Eiders Somateria mollissima

Human disturbance of nesting birds may cause reduced breeding success. It is therefore necessary to assess the impact of disturbance to identify steps that minimize negative impacts. We carried out a study of nesting success at two adjacent colonies of Common Eider Somateria mollissima on the islands of Grindøya and Håkøya in northern Norway between 2006 and 2011. Over the study period, nesting success was consistently higher on Håkøya (69–82%) than on Grindøya (35–60%). Between 2009 and 2011 we used camera monitoring of individual nests to identify determinants of nest survival and predation, focusing in particular on the effect of departures from the nest due to human disturbance, which differed between the colonies due to a long‐term research project on Grindøya. Overall, absence of Common Eiders from nests due to disturbance increased the predation risk by a factor of 6.42 for an increase of one additional daily disturbance. In contrast, absence due to natural recesses did not increase nest losses. Under high levels of human disturbance, camera monitoring indicated that the main cause of breeding failure was predation, primarily by Hooded Crows Corvus cornix, but also to some extent Great Black‐backed Gulls Larus marinus. The presence of cameras did not increase the predation risk. Both the presence of researchers and the sight of Common Eider females conspicuously departing from nests are likely to have provided cues to these predators. We suggest management trials to reduce nesting disturbance through the guarding of unoccupied nests to mitigate the effects of human disturbance on reproductive success.     

The striking polyphyly of Suiriri: Convergent evolution and social mimicry in two cryptic Neotropical birds

Two species of Suiriri (Aves: Tyrannidae) inhabit semi‐open habitats in South America: the polytypic Suiriri Flycatcher (S. suiriri) and the monotypic Chapada Flycatcher (S. affinis). The phylogenetic relationship between these congeneric species has never been investigated in detail. Here we used molecular tools—three nuclear introns and two mitochondrial genes—to investigate the systematic position of the Chapada Flycatcher, comparing the results found with morphological and behavioral data. We found that the polytypic Suiriri Flycatcher to be monophyletic and that it is included in a clade of Elaeniini flycatchers including Phyllomyias, Phaeomyias, and Capsiempis among other genera. The Chapada Flycatcher, on the other hand, is a member of the Fluvicolini, sister to Sublegatus, and should be allocated on its own monospecific genus, which we herein describe. We suggest that social mimicry is responsible for the remarkable convergence in size, shape, plumage coloration, and behavior in the adults of the Suiriri Flycatcher and the Chapada Flycatcher.     

Next‐generation DNA barcoding: using next‐generation sequencing to enhance and accelerate DNA barcode capture from single specimens

DNA barcoding is an efficient method to identify specimens and to detect undescribed/cryptic species. Sanger sequencing of individual specimens is the standard approach in generating large‐scale DNA barcode libraries and identifying unknowns. However, the Sanger sequencing technology is, in some respects, inferior to next‐generation sequencers, which are capable of producing millions of sequence reads simultaneously. Additionally, direct Sanger sequencing of DNA barcode amplicons, as practiced in most DNA barcoding procedures, is hampered by the need for relatively high‐target amplicon yield, coamplification of nuclear mitochondrial pseudogenes, confusion with sequences from intracellular endosymbiotic bacteria (e.g. Wolbachia) and instances of intraindividual variability (i.e. heteroplasmy). Any of these situations can lead to failed Sanger sequencing attempts or ambiguity of the generated DNA barcodes. Here, we demonstrate the potential application of next‐generation sequencing platforms for parallel acquisition of DNA barcode sequences from hundreds of specimens simultaneously. To facilitate retrieval of sequences obtained from individual specimens, we tag individual specimens during PCR amplification using unique 10‐mer oligonucleotides attached to DNA barcoding PCR primers. We employ 454 pyrosequencing to recover full‐length DNA barcodes of 190 specimens using 12.5% capacity of a 454 sequencing run (i.e. two lanes of a 16 lane run). We obtained an average of 143 sequence reads for each individual specimen. The sequences produced are full‐length DNA barcodes for all but one of the included specimens. In a subset of samples, we also detected Wolbachia, nontarget species, and heteroplasmic sequences. Next‐generation sequencing is of great value because of its protocol simplicity, greatly reduced cost per barcode read, faster throughout and added information content.     

Deceptive single‐locus taxonomy and phylogeography: Wolbachia‐associated divergence in mitochondrial DNA is not reflected in morphology and nuclear markers in a butterfly species

The satyrine butterfly Coenonympha tullia (Nymphalidae: Satyrinae) displays a deep split between two mitochondrial clades, one restricted to northern Alberta, Canada, and the other found throughout Alberta and across North America. We confirm this deep divide and test hypotheses explaining its phylogeographic structure. Neither genitalia morphology nor nuclear gene sequence supports cryptic species as an explanation, instead indicating differences between nuclear and mitochondrial genome histories. Sex‐biased dispersal is unlikely to cause such mito‐nuclear differences; however, selective sweeps by reproductive parasites could have led to this conflict. About half of the tested samples were infected by Wolbachia bacteria. Using multilocus strain typing for three Wolbachia genes, we show that the divergent mitochondrial clades are associated with two different Wolbachia strains, supporting the hypothesis that the mito‐nuclear differences resulted from selection on the mitochondrial genome due to selective sweeps by Wolbachia strains.     

Methodology to Improve the Efficiency of Evaluation of the Severity of Angular Leaf Spot in Common Bean

Disease severity assessment by means of a scoring scale, especially for angular leaf spot (Pseudocercospora griseola) in common bean, is hindered in experiments for assessment of progenies and/or breeding lines due to lack of uniformity of occurrence of the pathogens and segregation within progenies. The purpose of this study was to estimate the efficiency of the use of one plant per plot in assessing the severity of angular leaf spot in experiments for assessment of progenies and/or breeding lines in the common bean crop. To that end, two experimental strategies were used – one of them using one plant per plot and another using a standard size plot (SPP) (2–4‐m length rows). The experiments were conducted in the period from November 2011 to May 2012 in the municipalities of Lavras and Lambari, state of Minas Gerais, Brazil. Forty‐one lines from the breeding programme of the Universidade Federal de Lavras (UFLA) and from other research institutions were assessed, which differed in regard to their degree of susceptibility to P. griseola. The lines were assessed in regard to the severity of said disease using a five‐degree diagrammatic scale. In all the one plant per plot experiments, severity scores of angular leaf spot from the beginning of its occurrence, and later in intervals ranging from 7 to 12 days, were obtained. In the experiment with the SPP, assessment was made a few days prior to grain harvest. Estimates of the correlations between severity scores and grain yield (GY) were mostly of small magnitude. There was good coincidence between the lines classified as more resistant or more susceptible to the pathogen under the two conditions.     

Classification of crucigenoid algae: phylogenetic position of the reinstated genus Lemmermannia,Tetrastrum spp. Crucigenia tetrapedia,and C. lauterbornii (Trebouxiophyceae,Chlorophyta)1

The subfamily Crucigenioideae was traditionally classified within the well‐characterized family Scenedesmaceae (Chlorophyceae). Several morpho‐logical revisions and questionable taxonomic changes hampered the correct classification of crucigenoid species resulting in a high number of synonymous genera. We used a molecular approach to determine the phylogenetic position of several Tetrastrum and Crucigenia species. The molecular results were correlated with morphological and ontogenetic characters. Phylogenetic analyses of the SSU rDNA gene resolved the position of Tetrastrum heteracanthum and T. staurogeniaeforme as a new lineage within the Oocystis clade of the Trebouxiophyceae. Crucigenia tetrapedia, T. triangulare, T. punctatum, and T. komarekii were shown to be closely related to Botryococcus (Trebouxiophyceae) and were transferred to Lemmer‐mannia. Crucigenia lauterbornii was not closely related to the other Crucigenia strains, but was recovered within the Chlorella clade of the Trebouxiophyceae.     

The Relative Contribution of Specialists and Generalists to Mistletoe Dispersal: Insights from a Neotropical Rain Forest

Mistletoes rely on birds for seed dispersal, but the presumed importance of mistletoe‐specialist frugivores has not been critically examined nor compared with generalist frugivores and opportunistic foragers. The contribution of these three groups was compared directly by quantifying bird visitation to fruiting mistletoe plants ( Oryctanthus occidentalis: Loranthaceae) at Barro Colorado Island, Panama, and by comparing these results with proportions calculated from other empirical studies of mistletoe visitation conducted elsewhere. After more than 100 h of timed watches, 23 bird species were recorded visiting eight heavily infected host trees ( Luehea seemannii: Tiliaceae). Eight of these species visited mistletoe, of which five (all tyrannids) consumed mistletoe fruit. Although two mistletoe specialist frugivores ( Tyrannulus elatus and Zimmerius vilissimus) removed most fruit (73%), more than a quarter was consumed by one generalist frugivore ( Mionectes oleagineus) and two opportunists ( Myiozetetes cayanensis and Myiozetetes similis). Post consumption behaviour varied: the specialists flew from mistletoe to mistletoe, the generalist rested in the subcanopy and understory, and the opportunists spent most time hawking insects and resting high in the canopy. Integrating these data with previous work, the dietary specialization, short gut passage rate and strict habitat preferences of mistletoe specialists suggests that their services relate primarily to intensification and contagious dispersal, while species with broader diets are more likely to visit uninfected trees and establish new infections. The presumed importance of mistletoe‐specialist frugivores was not supported and mistletoes are considered to be comparable to many other bird‐dispersed plants, relying on both specialist and generalist frugivores, while opportunists may be disproportionately important in long‐distance dispersal.