This study presents genetic evidence that whale sharks, Rhincodon typus, are comprised of at least two populations that rarely mix and is the first to document a population expansion. Relatively high genetic structure is found when comparing sharks from the Gulf of Mexico with sharks from the Indo-Pacific. If mixing occurs between the Indian and Atlantic Oceans, it is not sufficient to counter genetic drift. This suggests whale sharks are not all part of a single global metapopulation. The significant population expansion we found was indicated by both microsatellite and mitochondrial DNA. The expansion may have happened during the Holocene, when tropical species could expand their range due to sea-level rise, eliminating dispersal barriers and increasing plankton productivity. However, the historic trend of population increase may have reversed recently. Declines in genetic diversity are found for 6 consecutive years at Ningaloo Reef in Australia. The declines in genetic diversity being seen now in Australia may be due to commercial-scale harvesting of whale sharks and collision with boats in past decades in other countries in the Indo-Pacific. The study findings have implications for models of population connectivity for whale sharks and advocate for continued focus on effective protection of the world's largest fish at multiple spatial scales.

Although the use of metabarcoding to identify taxa in DNA mixtures is widely approved, its reliability in quantifying taxon abundance is still the subject of debate. In this study we investigated the relationships between the amount of pollen grains in mock solutions and the abundance of highthroughput sequence reads and how the relationship was affected by the pollen counting methodology, the number of PCR cycles, the type of markers and plant species whose pollen grains have different characteristics. We found a significant positive relationship between the number of DNA sequences and the number of pollen grains in the mock solutions. However, better relationships were obtained with light microscopy as a pollen grain counting method compared with flow cytometry, with the chloroplastic trnL marker compared with ribosomal ITS1 and with 30 when compared with 25 or 35 PCR cycles. We provide a list of recommendations to improve pollen quantification. Environmental DNA metabarcoding is a molecular method that consists of investigating environmental DNA samples made of complex mixtures of genomes from numerous organisms 1. Due to new sequencing technologies and bioinformatics tools, metabarcoding has been increasingly used to identify taxa in environmental samples 1 to monitor biodiversity 2-4 , to investigate ecosystem functioning 5 and interaction networks 6-8 , in both aquatic and terrestrial ecosystems. Nevertheless, its reliability in quantitative approaches, which depend on the match between counts of high-throughput sequence reads and the amount of sampled biological material 2 , is still the subject of debate 9,10. While taxon identification can reveal individual diet breadth 11 , species richness, and the composition of habitats 2 , communities 12 and ecological networks 4 , taxon quantification provides knowledge on species evenness in those habitats, communities and diets or on the level of individual or species specialization in networks, all of which is very useful in ecological studies. Research on pollination and knowledge of the quantities of pollen transported by pollinators allow for the estimation of plant-pollinator interaction strength and hence it gives a more realistic representations of networks than those made possible using traditional approaches such as observing visits to plants by pollinators 9,13. Metabarcoding has been used in pollen studies to identify pollen in honey 14-16 , insect loads 6-8,17 , insect nests 18 , airborne samples 19 , and to quantify pollen abundance across various sample types. Several studies found significant positive relationships between pollen abundance (estimated using light microscopy) or pollen DNA quantities, and the abundance or the frequencies of high-throughput sequencing reads in experimental samples 10,16,17,20,21 , airborne samples 22,23 , insect pollen loads 21,24-27 or in brood cells of solitary bees 28. Conversely, other studies found low or no significant pollen-sequence abundance relationships when using ITS2 markers applied to pollen provision in...

Understanding the patterns of connectivity is required by the Strategic Plan for Biodiversity 2011-2020 and will be used to guide the extension of marine protection measures. Despite the increasing accuracy of ocean circulation modeling, the capacity to forecast the population connectivity of sessile benthic species with dispersal larval stages can be limited due to the potential effect of demographic filters acting before or after dispersal, which modulate offspring release or settlement, respectively. We applied an interdisciplinary approach that combined demographic surveys, genetic methods (assignment tests and coalescent-based analyses) and larval transport simulations to test the relative importance of demographics and ocean currents in shaping the contemporary patterns of gene flow among populations of a Mediterranean gorgonian (Eunicella singularis) in a fragmented rocky habitat (Gulf of Lion, NW Mediterranean Sea). We show that larval transport is a dominant driver of gene flow among the populations, and significant correlations were found between the contemporary gene flow and recent larval transport when the pelagic larval durations (PLDs) ranged from 7 to 14 days. Our results suggest that PLDs that efficiently connect populations distributed over a fragmented habitat are filtered by the habitat layout within the species competency period. Moreover, a PLD ranging from 7 to 14 days is sufficient to connect the fragmented rocky substrate of the Gulf of Lion. The rocky areas located in the center of the Gulf of Lion, which are currently not protected, were identified as essential hubs for the distribution of migrants in the region. We encourage the use of a range of PLDs instead of a single value when estimating larval transport with biophysical models to identify potential connectivity patterns among a network of marine protected areas or even solely a seascape. https://www.cbd.int/sp/targets/rationale/target-11/). Hence, understanding the degree and patterns of connectivity among existing MPAs and areas where protection could be extended becomes urgent. Connectivity is also known as the mechanism through which populations of sessile marine species, and particularly coral species, can recover after local and global disturbances (Roberts 1997;Hanski 1998;Cowen et al. 2000). In the context of global change, relating contemporary connectivity to the seascape processes that shape the spatial distribution of marine species is essential for the design of effective biodiversity conservation strategies. Seascape processes that drive population connectivity of sessile species with a larval dispersal stage, such as corals or gorgonians, should include both seabed geomorphology and dynamic hydrography (Manderson 2016). Correlations at the local scale between demographic population descriptors and the environment may, indeed, not be sufficient, and the successful exchange of individuals between distant locations (i.e., connectivity) should be included. Recent studies on seascape connectivity have focused on evaluati...