Pollen metabarcoding reveals the origin and multigenerational migratory pathway of an intercontinental-scale butterfly outbreak.

Migratory insects may move in large numbers, even surpassing migratory vertebrates in biomass. Long-distance migratory insects complete annual cycles through multiple generations, with each generation's reproductive success linked to the resources available at different breeding grounds. Climatic anomalies in these grounds are presumed to trigger rapid population outbreaks. Here, we infer the origin and track the multigenerational path of a remarkable outbreak of painted lady (Vanessa cardui) butterflies that took place at an intercontinental scale in Europe, the Middle East, and Africa from March 2019 to November 2019. Using metabarcoding, we identified pollen transported by 264 butterflies captured in 10 countries over 7 months and modeled the distribution of the 398 plants detected. The analysis showed that swarms collected in Eastern Europe in early spring originated in Arabia and the Middle East, coinciding with a positive anomaly in vegetation growth in the region from November 2018 to April 2019. From there, the swarms advanced to Northern Europe during late spring, followed by an early reversal toward southwestern Europe in summer. The pollen-based evidence matched spatiotemporal abundance peaks revealed by citizen science, which also suggested an echo effect of the outbreak in West Africa during September-November. Our results show that population outbreaks in a part of species' migratory ranges may disseminate demographic effects across multiple generations in a wide geographic area. This study represents an unprecedented effort to track a continuous multigenerational insect migration on an intercontinental scale.

Impact of the climatic changes in the Pliocene-Pleistocene transition on Irano-Turanian species. The radiation of genus Jurinea (Compositae).

The Irano-Turanian region is one of the world's richest floristic regions and the centre of diversity for numerous xerophytic plant lineages. However, we still have limited knowledge on the timing of evolution and biogeographic history of its flora, and potential drivers of diversification remain underexplored. To fill this knowledge gap, we focus on the Eurasian genus Jurinea (ca. 200 species), one of the largest plant radiations that diversified in the region. We applied a macroevolutionary integrative approach to explicitly test diversification hypotheses and investigate the relative roles of geography vs. ecology and niche conservatism vs. niche lability in speciation processes. To do so, we gathered a sample comprising 77% of total genus richness and obtained data about (1) its phylogenetic history, recovering 502 nuclear loci sequences; (2) growth forms; (3) ecological niche, compiling data of 21 variables for more than 2500 occurrences; and (4) paleoclimatic conditions, to estimate climatic stability. Our results revealed that climate was a key factor in the evolutionary dynamics of Jurinea. The main diversification and biogeographic events that occurred during past climate changes, which led to colder and drier conditions, are the following: (1) the origin of the genus (10.7 Ma); (2) long-distance dispersals from the Iranian Plateau to adjacent regions (∼7-4 Ma); and (3) the diversification shift during Pliocene-Pleistocene Transition (ca. 3 Ma), when net diversification rate almost doubled. Our results supported the pre-adaptation hypothesis, i.e., the evolutionary success of Jurinea was linked to the retention of the ancestral niche adapted to aridity. Interestingly, the paleoclimatic analyses revealed that in the Iranian Plateau long-term climatic stability favoured old-lineage persistence, resulting in current high species richness of semi-arid and cold adapted clades; whereas moderate climate oscillations stimulated allopatric diversification in the lineages distributed in the Circumboreal region. In contrast, growth form lability and high niche disparity among closely related species in the Central Asian clade suggest adaptive radiation to mountain habitats. In sum, the radiation of Jurinea is the result of both adaptive and non-adaptive processes influenced by climatic, orogenic and ecological factors.

Are Mediterranean Island Mountains Hotspots of Taxonomic and Phylogenetic Biodiversity? The Case of the Endemic Flora of the Balearic Islands.

The Mediterranean islands are exceptionally rich in endemism, most of which is narrowly distributed. Conservation measures, such as protected areas, have been prioritised, mainly on the basis of species richness and endemism, but phylogenetic information should also be taken into account. In this study, we calculated several taxonomic and phylogenetic metrics at a high resolution for the endemic flora of the Balearic Islands (154 taxa), in order to identify (i) the spatial patterns and environmental factors that explain this endemism, (ii) hotspots of species and phylogenetic endemism, and (iii) gaps in the protected areas. The taxonomic and phylogenetic metrics showed different distribution patterns, but the mountainous areas of Mallorca, and some coastal areas of the Balearic Islands, have the highest values. These values were positively related to elevation, precipitation, temperature, and slope, and negatively related to the distance from the coast, aspect, and the temperature of the wettest quarter. We identified top grid hotspots where all the metrics had the highest values, and we also identified nano-hotspots within these hotspots, in some of the highest peaks of Mallorca, where most of these metrics' maximum values coincided. This approach allowed us to identify some gaps in the conservation priority areas, and to highlight the need to review their boundaries and definition.

A Morphometric Analysis of the Santolina chamaecyparissus Complex (Asteraceae).

The genus Santolina (Asteraceae, Anthemideae) includes 26 species of aromatic evergreen shrubs endemic to the western Mediterranean Basin. Santolina is widely used as ornamental plant, in xerigardening, and in ethnobotany. The Santolina chamaecyparissus complex, including about half of the known species diversity, has been properly investigated on systematic and taxonomic grounds only recently, and a complete morphometric study is still missing. Here we provide a morphometric characterization and comparison of all the 14 species of this complex, using both univariate and multivariate analyses. Our results suggest that species of this complex can be distinguished using combinations of quantitative and qualitative character-states, mostly related to the leaf morphology. The analysis of S. villosa, a tetraploid/hexaploid Spanish endemic, showed that the two cytotypes cannot be safely identified based on morphology. Coupling this evidence with available phylogenetic information, we conclude that there is no reason to split the two cytotypes of S. villosa in two distinct taxa. An identification key for all the species of the complex is presented.

Re-establishment of Sileneneglecta Ten. (Caryophyllaceae) with taxonomic notes on some related taxa.

Sileneneglecta has been misunderstood and confused with S.nocturna, although several morphological characters (petal shape, calyx indumentum, hairiness of stamen filaments, seed size, seed-coat surface and shape) allow separation of these species. Moreover, S.mutabilis (which has been considered conspecific with S.neglecta) and S.martinolii (an alleged endemic species to south-western Sardinia) are considered here as taxonomic synonyms of S.nocturna and S.neglecta, respectively. These taxonomic conclusions are strongly supported by multivariate morphometric analyses of 21 characters.

On the identity of Thymushumifususvar.aureopunctatus (Lamiaceae) and taxonomic notes on the Th.richardii complex.

The name Thymushumifususvar.aureopunctatus, described from Bosnia and Herzegovina, is lectotypified, and its taxonomic value is discussed. Thymusrichardiisubsp.richardii is currently considered an endemic subspecies common to Mallorca (Balearic Islands) and Bosnia and Herzegovina from the Balkan Peninsula. Specimens identified as Th.richardii from both Balearic Islands and Bosnia and Herzegovina were studied to determine if they are indeed the same taxonomic entity. Detailed micromorphological observations and morphometric analysis, suggest that the Balkan plants (Th.humifususvar.aureopunctatus) and the Majorcan populations (Th.richardiisubsp.richardii) are clearly separate entities. For the former name, based on morphological, phytochemical, biogeographical and present results, we propose the subspecific rank, as Th.richardiisubsp.aureopunctatuscomb. & stat. nov. Full descriptions of all five subspecies currently accepted within Th.richardii are provided.

Nuclear and plastid DNA phylogeny of tribe Cardueae (Compositae) with Hyb-Seq data: A new subtribal classification and a temporal diversification framework.

Classification of tribe Cardueae in natural subtribes has always been a challenge due to the lack of support of some critical branches in previous phylogenies based on traditional Sanger markers. With the aim to propose a new subtribal delimitation, we applied a Hyb-Seq approach to a set of 76 Cardueae species representing all subtribes and informal groups defined in the tribe, targeting 1061 nuclear conserved orthology loci (COS) designed for Compositae and obtaining chloroplast coding regions as by-product of off-target reads. For the extraction of the target nuclear data, we used two strategies, PHYLUCE and HybPiper, and 776 and 1055 COS loci were recovered with each of them, respectively. Additionally, 87 chloroplast genes were assembled and annotated. With three datasets, phylogenetic relationships were reconstructed using both concatenation and coalescent approaches. Phylogenetic analyses of the nuclear datasets fully resolved virtually all nodes with very high support. Nuclear and plastid tree topologies are mostly congruent with a very limited number of incongruent nodes. Based on the well-solved phylogenies obtained, we propose a new taxonomic scheme of 12 monophyletic and morphologically consistent subtribes: Carlininae, Cardopatiinae, Echinopsinae, Dipterocominae (new), Xerantheminae (new), Berardiinae (new), Staehelininae (new), Onopordinae (new), Carduinae (redelimited), Arctiinae (new), Saussureinae (new), and Centaureinae. In addition, we further updated the temporal framework for origin and diversification of these subtribes. Our results highlight the power of Hyb-Seq over Sanger sequencing of a few DNA markers in solving phylogenetic relationships of traditionally difficult groups.

Pollen metabarcoding as a tool for tracking long-distance insect migrations.

Insects account for a large portion of Earth's biodiversity and are key players for ecosystems, notably as pollinators. While insect migration is suspected to represent a natural phenomenon of major importance, remarkably little is known about it, except for a few flagship species. The reason for this situation is mainly due to technical limitations in the study of insect movement. Here, we propose using metabarcoding of pollen carried by insects as a method for tracking their migrations. We developed a flexible and simple protocol allowing efficient multiplexing and not requiring DNA extraction, one of the most time-consuming part of metabarcoding protocols, and apply this method to the study of the long-distance migration of the butterfly Vanessa cardui, an emerging model for insect migration. We collected 47 butterfly samples along the Mediterranean coast of Spain in spring and performed metabarcoding of pollen collected from their bodies to test for potential arrivals from the African continent. In total, we detected 157 plant species from 23 orders, most of which (82.8%) were insect-pollinated. Taxa present in Africa-Arabia represented 73.2% of our data set, and 19.1% were endemic to this region, strongly supporting the hypothesis that migratory butterflies colonize southern Europe from Africa in spring. Moreover, our data suggest that a northwards trans-Saharan migration in spring is plausible for early arrivals (February) into Europe, as shown by the presence of Saharan floristic elements. Our results demonstrate the possibility of regular insect-mediated transcontinental pollination, with potential implications for ecosystem functioning, agriculture and plant phylogeography.

Phylogeography of western Mediterranean Cymbalaria (Plantaginaceae) reveals two independent long-distance dispersals and entails new taxonomic circumscriptions.

The Balearic Islands, Corsica and Sardinia (BCS) constitute biodiversity hotspots in the western Mediterranean Basin. Oligocene connections and long distance dispersal events have been suggested to cause presence of BCS shared endemic species. One of them is Cymbalaria aequitriloba, which, together with three additional species, constitute a polyploid clade endemic to BCS. Combining amplified fragment length polymorphism (AFLP) fingerprinting, plastid DNA sequences and morphometrics, we inferred the phylogeography of the group and evaluated the species' current taxonomic circumscriptions. Based on morphometric and AFLP data we propose a new circumscription for C. fragilis to additionally comprise a group of populations with intermediate morphological characters previously included in C. aequitriloba. Consequently, we suggest to change the IUCN category of C. fragilis from critically endangered (CR) to near threatened (NT). Both morphology and AFLP data support the current taxonomy of the single island endemics C. hepaticifolia and C. muelleri. The four species had a common origin in Corsica-Sardinia, and two long-distance dispersal events to the Balearic Islands were inferred. Finally, plastid DNA data suggest that interspecific gene flow took place where two species co-occur.

Resolving Recent Plant Radiations: Power and Robustness of Genotyping-by-Sequencing.

Disentangling species boundaries and phylogenetic relationships within recent evolutionary radiations is a challenge due to the poor morphological differentiation and low genetic divergence between species, frequently accompanied by phenotypic convergence, interspecific gene flow and incomplete lineage sorting. Here we employed a genotyping-by-sequencing (GBS) approach, in combination with morphometric analyses, to investigate a small western Mediterranean clade in the flowering plant genus Linaria that radiated in the Quaternary. After confirming the morphological and genetic distinctness of eight species, we evaluated the relative performances of concatenation and coalescent methods to resolve phylogenetic relationships. Specifically, we focused on assessing the robustness of both approaches to variations in the parameter used to estimate sequence homology (clustering threshold). Concatenation analyses suffered from strong systematic bias, as revealed by the high statistical support for multiple alternative topologies depending on clustering threshold values. By contrast, topologies produced by two coalescent-based methods (NJ$_{\mathrm{st}}$, SVDquartets) were robust to variations in the clustering threshold. Reticulate evolution may partly explain incongruences between NJ$_{\mathrm{st}}$, SVDquartets and concatenated trees. Integration of morphometric and coalescent-based phylogenetic results revealed (i) extensive morphological divergence associated with recent splits between geographically close or sympatric sister species and (ii) morphological convergence in geographically disjunct species. These patterns are particularly true for floral traits related to pollinator specialization, including nectar spur length, tube width and corolla color, suggesting pollinator-driven diversification. Given its relatively simple and inexpensive implementation, GBS is a promising technique for the phylogenetic and systematic study of recent radiations, but care must be taken to evaluate the robustness of results to variation of data assembly parameters.

Characterization of Zinc and Cadmium Hyperaccumulation in Three Noccaea (Brassicaceae) Populations from Non-metalliferous Sites in the Eastern Pyrenees.

The Southern slope of the Pyrenees is the meridional limit for the distribution of several Noccaea populations. However, the systematic description of these populations and their hyperaccumulation mechanisms are not well established. Morphological and genetic analysis (ITS and 3 chloroplast regions) were used to identify Noccaea populations localized on non-metallicolous soils during a survey in the Catalonian Pyrenees. Cd and Zn concentrations were analyzed in soils and plants both sampled in the field and grown hydroponically. The expression of selected metal transporter genes was assessed by quantitative PCR. The populations were identified as Noccaea brachypetala (Jord.) F.K. Mey by conspicuous morphological traits. Principal component analysis provided a clear separation among N. brachypetala, Noccaea caerulescens J. Presl & C. Presl and Noccaea occitanica (Jord.) F.K. Mey., three Noccaea species reported in the Pyrenees. Contrastingly, ITS and cpDNA analyses were unable to clearly differentiate these taxa. Differences in the expression of the metal transporter genes HMA3, HMA4, and MTP1 between N. caerulescens and N. brachypetala, and those amongst the N. brachypetala populations suggest differences in the strategies for handling enhanced Cd and Zn availability. This is the first report demonstrating Cd and Zn hyperaccumulation by N. brachypetala both in the field and in hydroponics. This comprehensive study based on taxonomic, molecular, and physiological data allows both the correct identification of this species and the characterization of population differences in hyperaccumulation and tolerance of Zn and Cd.

Genetic diversity of mountain plants: two migration episodes of Mediterranean Erodium (Geraniaceae).

This paper examines the phylogeny of Erodium subsect. Petraea, a group of six morphologically and genetically very similar species from the mountains of the western Mediterranean. Combined trnL-F-ITS analysis was unable to determine the phylogenetic relationships of these species owing to sequence similarity. AFLP fragment analysis showed different populations to cluster in six closely related phylogroups that partially coincided with morphological species. In the Iberian Peninsula, high temperatures during interstadial periods probably impeded the survival of these species at low altitudes, and their populations may have been forced to migrate northward within Iberia or remain isolated on high mountains. AFLP variation suggests that this might have led to their differentiation into groups and speciation during interglacials, but it probably also provided the basis for recurrent recolonisations and the mixing of neighbouring populations at the last glacial maxima. The genetic diversity of the two Erodium lineages suggests two migration episodes took place from southern Iberia towards the north, with one lineage migrating via western Iberia and the other via eastern Iberia. The patterns of genetic diversity observed in populations of 56 European species (27 genera) leads to the hypothesis that disparate proportions of unique polymorphic fragments are the result of the evolutionary histories of their mountain populations irrespective of the currently recognised species.

Reconstructing the history of Campanulaceae with a Bayesian approach to molecular dating and dispersal-vicariance analyses.

We reconstruct here the spatial and temporal evolution of the Campanula alliance in order to better understand its evolutionary history. To increase phylogenetic resolution among major groups (Wahlenbergieae-Campanuleae), new sequences from the rbcL region were added to the trnL-F dataset obtained in a previous study. These phylogenies were used to infer ancestral areas and divergence times in Campanula and related genera using a Bayesian approach to molecular dating and dispersal-vicariance analyses that takes into account phylogenetic uncertainty. The new phylogenetic analysis confirms Platycodoneae as the sister group of Wahlenbergieae-Campanuleae, the two last ones inter-graded into a well-supported clade. Biogeographic and dating analyses suggest that Western Asia and the Eastern Mediterranean have played a major role as centers of migration and diversification within the Campanula alliance, probably in relation to the intense orogenic activity that took place in this region during the Late Neogene, and that could have promoted isolation and allopatric speciation within lineages. Diversification rates within several Campanula lineages would have increased at the end of the Miocene, coinciding with the Messinian Stage. Strong selective pressures from climate changes and the expansion of mountainous regions during this period are suggested to explain the adaptation to drought, cold or disturbed environments observed in many Campanula species. Several independent long-distance dispersal events to North America are inferred within the Rapunculus clade, which seem to be related to high ploidy levels.