Molecular approach to describing a seed‐based food web: the post‐dispersal granivore community of an invasive plant

Communities of post‐dispersal granivores can shape the density and dispersion of exotic plants and invasive weeds, yet plant ecologists have a limited perception of the relative trophic linkages between a seed species and members of its granivore community. Dandelion seeds marked with Rabbit IgG were disseminated into replicated plots in the recipient habitat (South Dakota) and the native range (Czech Republic). Arthropods were collected in pitfall traps, and their guts were searched for the protein marker using enzyme‐linked immunosorbent assay (ELISA). Seed dishes were placed in each plot, and dandelion seed removal rates were measured. The entire experiment was repeated five times over the dandelion flowering period. Gut analysis revealed that approximately 22% of specimens tested positive for the seed marker. A more diverse granivore community had trophic linkages to seeds than has been previously realized under field conditions. This community included taxa such as isopods, millipedes, weevils, rove beetles, and caterpillars, in addition to the traditionally recognized ants, crickets, and carabid beetles. Rarefaction and Chao analysis estimated approximately 16 and 27 species in the granivore communities of the Czech Republic and South Dakota, respectively. Synthesis: Generalist granivore communities are diverse and polyphagous, and are clearly important as a form of biotic resistance to invasive and weedy plants. These granivore communities can be managed to limit population growth of these pests.     

Spatiotemporal and demographic variation in the diet of New Zealand lesser short‐tailed bats (Mystacina tuberculata)

Variation in the diet of generalist insectivores can be affected by site‐specific traits including weather, habitat, and season, as well as demographic traits such as reproductive status and age. We used molecular methods to compare diets of three distinct New Zealand populations of lesser short‐tailed bats, Mystacina tuberculata. Summer diets were compared between a southern cold‐temperate (Eglinton) and a northern population (Puroera). Winter diets were compared between Pureora and a subtropical offshore island population (Hauturu). This also permitted seasonal diet comparisons within the Pureora population. Lepidoptera and Diptera accounted for >80% of MOTUs identified from fecal matter at each site/season. The proportion of orders represented within prey and the Simpson diversity index, differed between sites and seasons within the Pureora population. For the Pureora population, the value of the Simpson diversity index was higher in summer than winter and was higher in Pureora compared to Eglinton. Summer Eglinton samples revealed that juvenile diets appeared to be more diverse than other demographic groups. Lactating females had the lowest dietary diversity during summer in Pureora. In Hauturu, we found a significant negative relationship between mean ambient temperature and prey richness. Our data suggest that M. tuberculata incorporate a narrower diversity of terrestrial insects than previously reported. This provides novel insights into foraging behavior and ecological interactions within different habitats. Our study is the first from the Southern Hemisphere to use molecular techniques to examine spatiotemporal variation in the diet of a generalist insectivore that inhabits a contiguous range with several habitat types and climates.     

Existence of species complex largely reduced barcoding success for invasive species of Tephritidae: a case study in Bactrocera spp.

Fruit flies in the family Tephritidae are the economically important pests that have many species complexes. DNA barcoding has gradually been verified as an effective tool for identifying species in a wide range of taxonomic groups, and there are several publications on rapid and accurate identification of fruit flies based on this technique; however, comprehensive analyses of large and new taxa for the effectiveness of DNA barcoding for fruit flies identification have been rare. In this study, we evaluated the COI barcode sequences for the diagnosis of fruit flies using 1426 sequences for 73 species of Bactrocera distributed worldwide. Tree‐based [neighbour‐joining (NJ)]; distance‐based, such as Best Match (BM), Best Close Match (BCM) and Minimum Distance (MD); and character‐based methods were used to evaluate the barcoding success rates obtained with maintaining the species complex in the data set, treating a species complex as a single taxon unit, and removing the species complex. Our results indicate that the average divergence between species was 14.04% (0.00–25.16%), whereas within a species this was 0.81% (0.00–9.71%); the existence of species complexes largely reduced the barcoding success for Tephritidae, for example relatively low success rates (74.4% based on BM and BCM and 84.8% based on MD) were obtained when the sequences from species complexes were included in the analysis, whereas significantly higher success rates were achieved if the species complexes were treated as a single taxon or removed from the data set – BM (98.9%), BCM (98.5%) and MD (97.5%), or BM (98.1%), BCM (97.4%) and MD (98.2%).     

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.     

Undaria pinnatifida: A case study to highlight challenges in marine invasion ecology and management

Marine invasion ecology and management have progressed significantly over the last 30 years although many knowledge gaps and challenges remain. The kelp Undaria pinnatifida, or “Wakame,” has a global non‐native range and is considered one of the world's “worst” invasive species. Since its first recorded introduction in 1971, numerous studies have been conducted on its ecology, invasive characteristics, and impacts, yet a general consensus on the best approach to its management has not yet been reached. Here, we synthesize current understanding of this highly invasive species and adopt Undaria as a case study to highlight challenges in wider marine invasion ecology and management. Invasive species such as Undaria are likely to continue to spread and become conspicuous, prominent components of coastal marine communities. While in many cases, marine invasive species have detectable deleterious impacts on recipient communities, in many others their influence is often limited and location specific. Although not yet conclusive, Undaria may cause some ecological impact, but it does not appear to drive ecosystem change in most invaded regions. Targeted management actions have also had minimal success. Further research is needed before well‐considered, evidence‐based management decisions can be made. However, if Undaria was to become officially unmanaged in parts of its non‐native range, the presence of a highly productive, habitat former with commercial value and a broad ecological niche, could have significant economic and even environmental benefit. How science and policy reacts to the continued invasion of Undaria may influence how similar marine invasive species are handled in the future.     

Biodiversity of green algae covering artificial hard substrate surfaces in a suburban environment: a case study using molecular approaches

In Middle European suburban environments green algae often cover open surfaces of artificial hard substrates. Microscopy reveals the Apatococcus/Desmococcus morphotype predominant over smaller coccoid forms. Adverse conditions such as limited water availability connected with high PAR and UV irradiance may narrow the algal diversity to a few specialists in these subaerial habitats. We used rRNA gene cloning/sequencing from both DNA extracts of the biofilms without culturing as well as cultures, for the unambiguous determination of the algal composition and to assess the algal diversity more comprehensively. The culture independent approach revealed mainly just two genera (Apatococcus, Trebouxia) for all study sites and five molecular operational taxonomic units (OTUs) for a particular study site, which based on microscopic observation was the one with the highest morphological diversity. The culture approach, however, revealed seven additional OTUs from five genera (Chloroidium, Coccomyxa, Coenochloris, Pabia, Klebsormidium) and an unidentified trebouxiophyte lineage for that same site; only two OTUs were shared by both approaches. Two OTUs or species were recovered for which references have been isolated only from Antarctica so far. However, the internal transcribed spacer (ITS) sequence differences among them supported they are representing distinct populations of the same species. Within Apatococcus five clearly distinct groups of ITS sequences, each putatively representing a distinct species, were recovered with three or four such ITS types co‐occurring at the same study site. Except for the streptophyte Klebsormidium only members of Trebouxiophyceae were detected suggesting these algae may be particularly well‐adapted to subaerial habitats.     

A molecular tool to identify Anastatus parasitoids of the brown marmorated stink bug

Globally, Anastatus species (Hymenoptera: Eupelmidae) are associated with the invasive agricultural pest Halyomorpha halys (Stål) (Hemiptera: Pentatomidae). In Europe, the polyphagous Anastatus bifasciatus (Geoffroy) is the most prevalent native egg parasitoid on H. halys eggs and is currently being tested as a candidate for augmentative biological control. Anastatus bifasciatus frequently displays behavior without oviposition, and induces additional host mortality through oviposition damage and host feeding that is not measured with offspring emergence. This exacerbates accurate assessment of parasitism and host impact, which is crucial for efficacy evaluation as well as for pre‐ and post‐release risk assessment. To address this, a general Anastatus primer set amplifying a 318‐bp fragment within the barcoding region of the cytochrome oxidase I (COI) gene was developed. When challenged with DNA of three Anastatus species —A. bifasciatus, Anastatus japonicus Ashmead, and Anastatus sp.—, five scelionid parasitoid species that might be encountered in the same host environments and 11 pentatomid host species, only Anastatus DNA was successfully amplified. When applied to eggs of the target host, H. halys, and an exemplary non‐target host, Dendrolimus pini L. (Lepidoptera: Lasiocampidae), subjected to host feeding, no Anastatus amplicons were produced. Eggs of the two host species containing A. bifasciatus parasitoid stages, from 1‐h‐old eggs to pupae, and emerged eggs yielded Anastatus fragments. Confirmation of parasitoid presence with dissections and subsequent PCRs with the developed primer pair resulted in 95% success for 1‐h‐old parasitoid eggs. For both host species, field‐exposed sentinel emerged eggs stored dry for 6 months, 100% of the specimens produced Anastatus amplicons. This DNA‐based screening method can be used in combination with conventional methods to better interpret host‐parasitoid and parasitoid‐parasitoid interactions. It will help address ecological questions related to an environmentally friendly approach for the control of H. halys in invaded areas.     

A tough egg to crack: recreational boats as vectors for invasive goby eggs and transdisciplinary management approaches

Non‐native invasive species are a major threat to biodiversity, especially in freshwater ecosystems. Freshwater ecosystems are naturally rather isolated from one another. Nonetheless, invasive species often spread rapidly across water sheds. This spread is to a large extent realized by human activities that provide vectors. For example, recreational boats can carry invasive species propagules as “aquatic hitch‐hikers” within and across water sheds. We used invasive gobies in Switzerland as a case study to test the plausibility that recreational boats can serve as vectors for invasive fish and that fish eggs can serve as propagules. We found that the peak season of boat movements across Switzerland and the goby spawning season overlap temporally. It is thus plausible that goby eggs attached to boats, anchors, or gear may be transported across watersheds. In experimental trials, we found that goby eggs show resistance to physical removal (90 mN attachment strength of individual eggs) and stay attached if exposed to rapid water flow (2.8 m·s⁻¹for 1 h). When exposing the eggs to air, we found that hatching success remained high (>95%) even after eggs had been out of water for up to 24 h. It is thus plausible that eggs survive pick up, within‐water and overland transport by boats. We complemented the experimental plausibility tests with a survey on how decision makers from inside and outside academia rate the feasibility of managing recreational boats as vectors. We found consensus that an installation of a preventive boat vector management is considered an effective and urgent measure. This study advances our understanding of the potential of recreational boats to serve as vectors for invasive vertebrate species and demonstrates that preventive management of recreational boats is considered feasible by relevant decision makers inside and outside academia.     

Divergent demographic patterns and perspectives for conservation of endemic species in extreme environments: a case study of the springsnail Melanopsis etrusca (Gastropoda: Melanopsidae)

We studied the distribution, size structure, growth, and diet of the freshwater prosobranch Melanopsis etrusca in experimental and natural population samples. This snail is endemic to isolated headwaters of thermal springs in central Italy. In recent times, several anthropogenic pressures such as water extraction and stream contamination have caused population declines and local extinctions of M. etrusca. Consequently, this species was included on the IUCN Red List of Threatened Species. The streams inhabited by M. etrusca had oligohaline waters; however, some physicochemical variability existed among sites. We found that the two variables most directly related to the patterns of distribution and life history of M. etrusca were water pH and temperature. In particular, individual growth was faster in streams with water temperatures constantly >30°C, which was reflected by a shorter life cycle than in cooler streams (13°–26°C), where snails showed a seasonal pattern of growth. Finally, we observed that individuals in a population that is exposed to extreme temperatures (>35°C) performed a peculiar behavior, spending long periods outside the water. We also found that the diet of M. etrusca reflected the local composition of the periphytic community and was dominated by macrophyte fragments. Our data suggest that heat‐adapted populations of M. etrusca hold the greatest potential for recolonization after collapse events. From a conservation perspective, reintroduction programs could be useful to avoid local extinction or bottleneck effects but at the risk of the loss of evolutionary innovations that may have emerged at the population level.     

Using HHsearch to tackle proteins of unknown function: A pilot study with PH domains

Advances in membrane cell biology are hampered by the relatively high proportion of proteins with no known function. Such proteins are largely or entirely devoid of structurally significant domain annotations. Structural bioinformaticians have developed profile‐profile tools such as HHsearch (online version called HHpred), which can detect remote homologies that are missed by tools used to annotate databases. Here we have applied HHsearch to study a single structural fold in a single model organism as proof of principle. In the entire clan of protein domains sharing the pleckstrin homology domain fold in yeast, systematic application of HHsearch accurately identified known PH‐like domains. It also predicted 16 new domains in 13 yeast proteins many of which are implicated in intracellular traffic. One of these was Vps13p, where we confirmed the functional importance of the predicted PH‐like domain. Even though such predictions require considerable work to be corroborated, they are useful first steps. HHsearch should be applied more widely, particularly across entire proteomes of model organisms, to significantly improve database annotations.      

DNA metabarcoding for diet analysis and biodiversity: A case study using the endangered Australian sea lion (Neophoca cinerea)

The analysis of apex predator diet has the ability to deliver valuable insights into ecosystem health, and the potential impacts a predator might have on commercially relevant species. The Australian sea lion (Neophoca cinerea) is an endemic apex predator and one of the world's most endangered pinnipeds. Given that prey availability is vital to the survival of top predators, this study set out to understand what dietary information DNA metabarcoding could yield from 36 sea lion scats collected across 1,500 km of its distribution in southwest Western Australia. A combination of PCR assays were designed to target a variety of potential sea lion prey, including mammals, fish, crustaceans, cephalopods, and birds. Over 1.2 million metabarcodes identified six classes from three phyla, together representing over 80 taxa. The results confirm that the Australian sea lion is a wide‐ranging opportunistic predator that consumes an array of mainly demersal fauna. Further, the important commercial species Sepioteuthis australis (southern calamari squid) and Panulirus cygnus (western rock lobster) were detected, but were present in <25% of samples. Some of the taxa identified, such as fish, sharks and rays, clarify previous knowledge of sea lion prey, and some, such as eel taxa and two gastropod species, represent new dietary insights. Even with modest sample sizes, a spatial analysis of taxa and operational taxonomic units found within the scat shows significant differences in diet between many of the sample locations and identifies the primary taxa that are driving this variance. This study provides new insights into the diet of this endangered predator and confirms the efficacy of DNA metabarcoding of scat as a noninvasive tool to more broadly define regional biodiversity.     

Evidence for a recent horizontal transmission and spatial spread of Wolbachia from endemic Rhagoletis cerasi (Diptera: Tephritidae) to invasive Rhagoletis cingulata in Europe

The widespread occurrence of Wolbachia in arthropods and nematodes suggests that this intracellular, maternally inherited endosymbiont has the ability to cross species boundaries. However, direct evidence for such a horizontal transmission of Wolbachia in nature is scarce. Here, we compare the well‐characterized Wolbachia infection of the European cherry fruit fly, Rhagoletis cerasi, with that of the North American eastern cherry fruit fly, Rhagoletis cingulata, recently introduced to Europe. Molecular genetic analysis of Wolbachia based on multilocus sequence typing and the Wolbachia surface protein wsp showed that all R. cingulata individuals are infected with wCin2 identical to wCer2 in R. cerasi. In contrast, wCin1, a strain identical to wCer1 in R. cerasi, was present in several European populations of R. cingulata, but not in any individual from the United States. Surveys of R. cingulata from Germany and Hungary indicated that in some populations, the frequency of wCin1 increased significantly in just a few years with at least two independent horizontal transmission events. This is corroborated by the analysis of the mitochondrial cytochrome oxidase II gene that showed association of wCin1 with two distinct haplotypes in Germany, one of which is also infected with wCin1 in Hungary. In summary, our study provides strong evidence for a very recent inter‐specific Wolbachia transmission with a subsequent spatial spread in field populations.     

Corroborating trackmaker identification through footprint functional analysis: the case study of Ichniotherium and Dimetropus

A study carried out on some excellently preserved footprints has revealed the vital importance of functionality analysis, together with morphological analyses, in settling systematics and attribution issues in ichnological studies. To test the alleged reliability of functionality studies in tetrapod ichnology, a careful analysis of numerous specimens (both trackways and isolated footprints) of the Gotha collection, referred to the Permian ichnotaxa Ichniotherium and Dimetropus, has been carried out. The method, in addition to the classical ichnological protocol, includes a careful and basic analysis of the differential depth of the impression to evaluate biomechanics and functional potentiality of the putative trackmaker in the light of osteological structures. The analysis has led to the recognition of two well‐separated particular functionalities (over a complete locomotion cycle), sharply distinguishable in Ichniotherium and Dimetropus. The Ichniotherium trackmaker presents a functional axis in the fore and hind limbs moved always along the medial side during the stroke progression; in Dimetropus trackmaker there are at least two functional axes, the first moved laterally in the initial stroke phase and the second moved medially during the maximum load phase. Those different functionalities are intimately linked to the particular osteological characters present in diadectids and non‐therapsid synapsids. Thus, functionality analysis in the ichnological practice is a valuable tool strongly recommended for use whenever possible.     

Generalist trophic ecology in a changing habitat: The case of the four‐striped mouse in a woody‐encroached savannah

Trophic generalists tolerate greater habitat change than specialists; however, few studies explore how generalist trophic ecology is affected. We established how the trophic ecology of an extreme generalist, Rhabdomys pumilio, changed in relation to a directionally changing woody‐encroached savannah in Eastern Cape, South Africa by investigating (a) foraging behaviour, (b) trophic niche and (c) feedback effects. (a) Giving‐up densities showed that R. pumilio preferred foraging in subcanopy microhabitat during the night as a result of lower thermoregulatory costs, but had similar preferences for sub‐ and intercanopy microhabitats during the day. (b) An isotope analysis revealed that the dietary composition and trophic niche occupied by R. pumilio differed among tree canopy cover levels (0%, 30% and 80%), which appeared to be related to changes in C₄ grass material and invertebrate availability. (c) Artificial seed patches suggested that R. pumilio was a potentially important postdispersal seed predator of the woody‐encroaching species, Vachellia karroo. Thus, an increase in tree canopy cover altered the trophic niche of R. pumilio by reducing foraging costs at night and providing alternative food resources in terms of availability and source. These findings demonstrate how an extreme generalist adapted to human‐induced habitat change through changes in its trophic ecology.