Finding the perfect pairs: A matchmaking of plant markers and primers for multi-marker eDNA metabarcoding.

As the scope of plant eDNA metabarcoding diversifies, so do the primers, markers and methods. A wealth of primers exists today, but their comparative evaluation is lacking behind. Similarly, multi-marker approaches are recommended but debates persist regarding barcode complementarity and optimal combinations. After a literature compilation of used primers, we compared in silico 102 primer pairs based on amplicon size, coverage and specificity, followed by an experimental evaluation of 15 primer pairs on a mock community sample covering 268 plant species and genera, and about 100 families. The analysis was done for the four most common plant metabarcoding markers, rbcL, trnL, ITS1 and ITS2 and their complementarity was assessed based on retrieved species. By focusing on existing primers, we identify common designs, promote alternatives and enhance prior-supported primers for immediate applications. The ITS2 was the best-performing marker for flowering vascular plants and was congruent to ITS1. However, the combined taxonomic breadth of ITS2 and rbcL surpassed any other combination, highlighting their high complementarity across Streptophyta. Overall, our study underscores the significance of comprehensive primer and barcode evaluations tailored to metabarcoding applications.

How can a rare protected plant cope with the metal and metalloid soil pollution resulting from past industrial activities? Phytometabolites, antioxidant activities and root symbiosis involved in the metal tolerance of Astragalus tragacantha.

Astragalus tragacantha is a protected plant species in France that grows even in the trace metal and metalloid (TMM) polluted soils of the Calanques National Park (PNCal). Soils are mainly contaminated by lead, copper, zinc and arsenic. An ex situ experiment was conducted, firstly to determine the molecular responses and root traits involved in the TMM tolerance of this plant species by growing individuals in a soil from the surroundings of one of the brownfields of the PNCal, known as l'Escalette, where this plant species grows spontaneously. Secondly, in order to determine the plasticity of these responses, seeds were collected from three different populations, at l'Escalette (polluted site), one from the Frioul archipelago (non-polluted, insular site) and one from La Seyne (non-polluted, littoral site). The results of this study confirmed the capacity of A. tragacantha to germinate and grow in TMM contaminated soils. Only moderate significant variations in chlorophyll and flavonol indices, proline content and antioxidant activities were detected between polluted and control soil conditions for all populations. The main driver for A. tragacantha TMM tolerance seemed to be its ability to be associated with root symbionts i.e. arbuscular mycorrhizal fungi and dark septate endophytes, corresponding to a nutrient-uptake strategy trait. This work provides support for the challenge of A. tragacantha conservation along the littoral of the PNCal, because increasing the number of A. tragacantha individuals would both increase vegetation cover of the polluted soils to reduce the pollution transfer and reinforce the populations of this species.

A minimalist macroparasite diversity in the round goby of the Upper Rhine reduced to an exotic acanthocephalan lineage.

The round goby, Neogobius melanostomus, is a Ponto-Caspian fish considered as an invasive species in a wide range of aquatic ecosystems. To understand the role that parasites may play in its successful invasion across Western Europe, we investigated the parasitic diversity of the round goby along its invasion corridor, from the Danube to the Upper Rhine rivers, using data from literature and a molecular barcoding approach, respectively. Among 1666 parasites extracted from 179 gobies of the Upper Rhine, all of the 248 parasites barcoded on the c oxidase subunit I gene were identified as Pomphorhynchus laevis. This lack of macroparasite diversity was interpreted as a loss of parasites along its invasion corridor without spillback compensation. The genetic diversity of P. laevis was represented by 33 haplotypes corresponding to a haplotype diversity of 0·65 ± 0·032, but a weak nucleotide diversity of 0·0018 ± 0·00015. Eight of these haplotypes were found in 88·4% of the 248 parasites. These haplotypes belong to a single lineage so far restricted to the Danube, Vistula and Volga rivers (Eastern Europe). This result underlines the exotic status of this Ponto-Caspian lineage in the Upper Rhine, putatively disseminated by the round goby along its invasion corridor.

Species Distribution 2.0: An Accurate Time- and Cost-Effective Method of Prospection Using Street View Imagery.

Species occurrence data provide crucial information for biodiversity studies in the current context of global environmental changes. Such studies often rely on a limited number of occurrence data collected in the field and on pseudo-absences arbitrarily chosen within the study area, which reduces the value of these studies. To overcome this issue, we propose an alternative method of prospection using geo-located street view imagery (SVI). Following a standardised protocol of virtual prospection using both vertical (aerial photographs) and horizontal (SVI) perceptions, we have surveyed 1097 randomly selected cells across Spain (0.1x0.1 degree, i.e. 20% of Spain) for the presence of Arundo donax L. (Poaceae). In total we have detected A. donax in 345 cells, thus substantially expanding beyond the now two-centuries-old field-derived record, which described A. donax only 216 cells. Among the field occurrence cells, 81.1% were confirmed by SVI prospection to be consistent with species presence. In addition, we recorded, by SVI prospection, 752 absences, i.e. cells where A. donax was considered absent. We have also compared the outcomes of climatic niche modeling based on SVI data against those based on field data. Using generalized linear models fitted with bioclimatic predictors, we have found SVI data to provide far more compelling results in terms of niche modeling than does field data as classically used in SDM. This original, cost- and time-effective method provides the means to accurately locate highly visible taxa, reinforce absence data, and predict species distribution without long and expensive in situ prospection. At this time, the majority of available SVI data is restricted to human-disturbed environments that have road networks. However, SVI is becoming increasingly available in natural areas, which means the technique has considerable potential to become an important factor in future biodiversity studies.

Geographical isolation caused the diversification of the Mediterranean thorny cushion-like Astragalus L. sect. Tragacantha DC. (Fabaceae).

Understanding the origin and evolution of Mediterranean vascular flora within the long-term context of climate change requires a continuous study of historical biogeography supported by molecular phylogenetic approaches. Here we provide new insights into the fascinating but often overlooked diversification of Mediterranean xerophytic plants. Growing in some of the most stressing Mediterranean environments, i.e. coastal and mountainous opened habitats, the circum-Mediterranean Astragalus L. sect. Tragacantha DC. (Fabaceae) gathers several thorny cushion-like taxa. These have been the subjects of recent taxonomical studies, but they have not yet been investigated within a comprehensive molecular framework. Bayesian phylogenetics applied to rDNA ITS sequences reveal that the diversification of A. sect. Tragacantha has roots dating back to the Pliocene, and the same data also indicate an eastern-western split giving rise to the five main lineages that exist today. In addition, AFLP fingerprinting supports an old east-west pattern of vicariance that completely rules out the possibility of a recent eastern origin for western taxa. The observed network of genetic relationships implies that contrary to what is widely claimed in the taxonomic literature, it is range fragmentation, as opposed to a coastal-to-mountain ecological shift, that is likely the main driver of diversification.

Impact of polyploidy on fertility variation of Mediterranean Arundo L. (Poaceae).

Failure of seed production in the genus Arundo L. (Poaceae) is often attributed to polyploidy. This study tested the impact of two ploidy levels (2n=12 and 18x) on the fertility of four Mediterranean Arundo. Viable pollen was screened from its production to its germination, and seed occurrence was monitored in admixture or isolated conditions. In addition, insights on restructuration of polyploid genomes were analysed using molecular cytogenetics. Our results show that high ploidy levels do not automatically induce failure of sexual reproduction. The two ploidy levels are able to produce viable pollen and seed set depending on species and cultural conditions. The sterility of A. micrantha (2n=12x) and A. donax (2n=18x) is due to the early failures of gametogenesis steps. For 18x cytotypes of A. donaciformis and A. plinii, seed absence for isolated genotype vs. seed production in admixed culture support their auto-incompatibility.

Origin of the invasive Arundo donax (Poaceae): a trans-Asian expedition in herbaria.

BACKGROUND AND AIMS: The hypothesis of an ancient introduction, i.e. archaeophyte origin, is one of the most challenging questions in phylogeography. Arundo donax (Poaceae) is currently considered to be one of the worst invasive species globally, but it has also been widely utilzed by man across Eurasia for millennia. Despite a lack of phylogenetic data, recent literature has often speculated on its introduction to the Mediterranean region. METHODS: This study tests the hypothesis of its ancient introduction from Asia to the Mediterranean by using plastid DNA sequencing and morphometric analysis on 127 herbarium specimens collected across sub-tropical Eurasia. In addition, a bioclimatic species distribution model calibrated on 1221 Mediterranean localities was used to identify similar ecological niches in Asia. KEY RESULTS: Despite analysis of several plastid DNA hypervariable sites and the identification of 13 haplotypes, A. donax was represented by a single haplotype from the Mediterranean to the Middle East. This haplotype is shared with invasive samples worldwide, and its nearest phylogenetic relatives are located in the Middle East. Morphometric data characterized this invasive clone by a robust morphotype distinguishable from all other Asian samples. The ecological niche modelling designated the southern Caspian Sea, southern Iran and the Indus Valley as the most suitable regions of origin in Asia for the invasive clone of A. donax. CONCLUSIONS: Using an integrative approach, an ancient dispersion of this robust, polyploid and non-fruiting clone is hypothesized from the Middle East to the west, leading to its invasion throughout the Mediterranean Basin.