Contrasting new and available reference genomes to highlight uncertainties in assemblies and areas for future improvement: an example with monodontid species.

BACKGROUND: Reference genomes provide a foundational framework for evolutionary investigations, ecological analysis, and conservation science, yet uncertainties in the assembly of reference genomes are difficult to assess, and by extension rarely quantified. Reference genomes for monodontid cetaceans span a wide spectrum of data types and analytical approaches, providing the context to derive broader insights related to discrepancies and regions of uncertainty in reference genome assembly. We generated three beluga (Delphinapterus leucas) and one narwhal (Monodon monoceros) reference genomes and contrasted these with published chromosomal scale assemblies for each species to quantify discrepancies associated with genome assemblies. RESULTS: The new reference genomes achieved chromosomal scale assembly using a combination of PacBio long reads, Illumina short reads, and Hi-C scaffolding data. For beluga, we identified discrepancies in the order and orientation of contigs in 2.2-3.7% of the total genome depending on the pairwise comparison of references. In addition, unsupported higher order scaffolding was identified in published reference genomes. In contrast, we estimated 8.2% of the compared narwhal genomes featured discrepancies, with inversions being notably abundant (5.3%). Discrepancies were linked to repetitive elements in both species. CONCLUSIONS: We provide several new reference genomes for beluga (Delphinapterus leucas), while highlighting potential avenues for improvements. In particular, additional layers of data providing information on ultra-long genomic distances are needed to resolve persistent errors in reference genome construction. The comparative analyses of monodontid reference genomes suggested that the three new reference genomes for beluga are more accurate compared to the currently published reference genome, but that the new narwhal genome is less accurate than one published. We also present a conceptual summary for improving the accuracy of reference genomes with relevance to end-user needs and how they relate to levels of assembly quality and uncertainty.

Structure and formation of the perforated theca defining the Pelagophyceae (Heterokonta), and three new genera that substantiate the diverse nature of the class.

The pelagophytes, a morphologically diverse class of marine heterokont algae, have been historically united only by DNA sequences. Recently we described a novel perforated theca (PT) encasing cells from the Pelagophyceae and hypothesized it may be the first morphological feature to define the class. Here we consolidate that observation, describing a PT for the first time in an additional seven pelagophyte genera, including three genera new to science. We established clonal cultures of pelagophytes collected from intertidal pools located around Australia, and established phylogenetic trees based on nuclear 18S rDNA and plastid rbcL, psaA, psaB, psbA and psbC gene sequences that led to the discovery of three new species: Wyeophycus julieharrissiae and Chromopallida australis form a distinct lineage along with Ankylochrysis lutea within the Pelagomonadales, while Pituiglomerulus capricornicus is sister genus to Chrysocystis fragilis in the Chrysocystaceae (Sarcinochrysidales). Using fixation by high-pressure freezing for electron microscope observations, a distinctive PT was observed in the three new genera described in this paper, as well as four genera not previously investigated: Chrysoreinhardia, Sargassococcus, Sungminbooa and Andersenia. The mechanism of PT formation is novel, being fabricated from rafts in Golgi-derived vesicles before being inserted into an established PT. Extracellular wall and/or mucilage layers assemble exterior to the PT in most pelagophytes, the materials likewise secreted by Golgi-derived vesicles, though the mechanism of secretion is novel. Secretory vesicles never fuse with the plasma membrane as in classic secretion and deposition, but rather relocate extracellularly beneath the PT and disintegrate, the contents having to pass through the PT prior to wall and/or mucilage synthesis. This study substantiates the diverse nature of pelagophytes, and provides further evidence that the PT is a sound morphological feature to define the Pelagophyceae, with all 14 of the 20 known genera studied to date by TEM possessing a PT.

Whole genome population structure of North Atlantic kelp confirms high-latitude glacial refugia.

Coastal refugia during the Last Glacial Maximum (~21,000 years ago) have been hypothesized at high latitudes in the North Atlantic, suggesting marine populations persisted through cycles of glaciation and are potentially adapted to local environments. Here, whole-genome sequencing was used to test whether North Atlantic marine coastal populations of the kelp Alaria esculenta survived in the area of southwestern Greenland during the Last Glacial Maximum. We present the first annotated genome for A. esculenta and call variant positions in 54 individuals from populations in Atlantic Canada, Greenland, Faroe Islands, Norway and Ireland. Differentiation across populations was reflected in ~1.9 million single nucleotide polymorphisms, which further revealed mixed ancestry in the Faroe Islands individuals between putative Greenlandic and European lineages. Time-calibrated organellar phylogenies suggested Greenlandic populations were established during the last interglacial period more than 100,000 years ago, and that the Faroe Islands population was probably established following the Last Glacial Maximum. Patterns in population statistics, including nucleotide diversity, minor allele frequencies, heterozygosity and linkage disequilibrium decay, nonetheless suggested glaciation reduced Canadian Atlantic and Greenlandic populations to small effective sizes during the most recent glaciation. Functional differentiation was further reflected in exon read coverage, which revealed expansions unique to Greenland in 337 exons representing 162 genes, and a modest degree of exon loss (103 exons from 56 genes). Altogether, our genomic results provide strong evidence that A. esculenta populations were resilient to past climatic fluctuations related to glaciations and that high-latitude populations are potentially already adapted to local conditions as a result.

Arctic marine forest distribution models showcase potentially severe habitat losses for cryophilic species under climate change.

The Arctic is among the fastest-warming areas of the globe. Understanding the impact of climate change on foundational Arctic marine species is needed to provide insight on ecological resilience at high latitudes. Marine forests, the underwater seascapes formed by seaweeds, are predicted to expand their ranges further north in the Arctic in a warmer climate. Here, we investigated whether northern habitat gains will compensate for losses at the southern range edge by modelling marine forest distributions according to three distribution categories: cryophilic (species restricted to the Arctic environment), cryotolerant (species with broad environmental preferences inclusive but not limited to the Arctic environment), and cryophobic (species restricted to temperate conditions) marine forests. Using stacked MaxEnt models, we predicted the current extent of suitable habitat for contemporary and future marine forests under Representative Concentration Pathway Scenarios of increasing emissions (2.6, 4.5, 6.0, and 8.5). Our analyses indicate that cryophilic marine forests are already ubiquitous in the north, and thus cannot expand their range under climate change, resulting in an overall loss of habitat due to severe southern range contractions. The extent of marine forests within the Arctic basin, however, is predicted to remain largely stable under climate change with notable exceptions in some areas, particularly in the Canadian Archipelago. Succession may occur where cryophilic and cryotolerant species are extirpated at their southern range edge, resulting in ecosystem shifts towards temperate regimes at mid to high latitudes, though many aspects of these shifts, such as total biomass and depth range, remain to be field validated. Our results provide the first global synthesis of predicted changes to pan-Arctic coastal marine forest ecosystems under climate change and suggest ecosystem transitions are unavoidable now for some areas.

Whole-genome sequencing reveals forgotten lineages and recurrent hybridizations within the kelp genus Alaria (Phaeophyceae).

The genomic era continues to revolutionize our understanding of the evolution of biodiversity. In phycology, emphasis remains on assembling nuclear and organellar genomes, leaving the full potential of genomic datasets to answer long-standing questions about the evolution of biodiversity largely unexplored. Here, we used whole-genome sequencing (WGS) datasets to survey species diversity in the kelp genus Alaria, compare phylogenetic signals across organellar and nuclear genomes, and specifically test whether phylogenies behave like trees or networks. Genomes were sequenced from across the global distribution of Alaria (including Alaria crassifolia, A. praelonga, A. crispa, A. marginata, and A. esculenta), representing over 550 GB of data and over 2.2 billion paired reads. Genomic datasets retrieved 3,814 and 4,536 single-nucleotide polymorphisms (SNPs) for mitochondrial and chloroplast genomes, respectively, and upwards of 148,542 high-quality nuclear SNPs. WGS revealed an Arctic lineage of Alaria, which we hypothesize represents the synonymized taxon A. grandifolia. The SNP datasets also revealed inconsistent topologies across genomic compartments, and hybridization (i.e., phylogenetic networks) between Pacific A. praelonga, A. crispa, and putative A. grandifolia, and between some lineages of the A. marginata complex. Our analysis demonstrates the potential for WGS data to advance our understanding of evolution and biodiversity beyond amplicon sequencing, and that hybridization is potentially an important mechanism contributing to novel lineages within Alaria. We also emphasize the importance of surveying phylogenetic signals across organellar and nuclear genomes, such that models of mixed ancestry become integrated into our evolutionary and taxonomic understanding.

Genomic Rearrangements and Sequence Evolution across Brown Algal Organelles.

Organellar genomes serve as useful models for genome evolution and contain some of the most widely used phylogenetic markers, but they are poorly characterized in many lineages. Here, we report 20 novel mitochondrial genomes and 16 novel plastid genomes from the brown algae. We focused our efforts on the orders Chordales and Laminariales but also provide the first plastid genomes (plastomes) from Desmarestiales and Sphacelariales, the first mitochondrial genome (mitome) from Ralfsiales and a nearly complete mitome from Sphacelariales. We then compared gene content, sequence evolution rates, shifts in genome structural arrangements, and intron distributions across lineages. We confirm that gene content is largely conserved in both organellar genomes across the brown algal tree of life, with few cases of gene gain or loss. We further show that substitution rates are generally lower in plastid than mitochondrial genes, but plastomes are more variable in gene arrangement, as mitomes tend to be colinear even among distantly related lineages (with exceptions). Patterns of intron distribution across organellar genomes are complex. In particular, the mitomes of several laminarialean species possess group II introns that have T7-like ORFs, found previously only in mitochondrial genomes of Pylaiella spp. (Ectocarpales). The distribution of these mitochondrial introns is inconsistent with vertical transmission and likely reflects invasion by horizontal gene transfer between lineages. In the most extreme case, the mitome of Hedophyllum nigripes is ∼40% larger than the mitomes of close relatives because of these introns. Our results provide substantial insight into organellar evolution across the brown algae.

New pelagophytes show a novel mode of algal colony development and reveal a perforated theca that may define the class.

Pelagophytes (Heterokonta) are a morphologically diverse class of marine algae historically united only by DNA sequences. We established clonal cultures of sand-dwelling pelagophytes collected from intertidal pools around Australia. Phylogenetic trees based on nuclear 18S rDNA and plastid rbcL, psaA, psaB, psbA, and psbC sequences revealed two new genera, Gazia and Glomerochrysis, related to Aureoumbra in a distinct lineage within the Sarcinochrysidaceae (Pelagophyceae). The three new species (Gazia saundersii, Gazia australica, and Glomerochrysis psammophila), along with an Australian strain of Aureoumbra geitleri, are characterized by dominant benthic stages that differ significantly from one another, while occasionally producing classic heterokont zoospores. The benthic stage of Ga. saundersii has a novel development not observed in any other colonial alga, consisting of large, spherical colonies (up to 140 µm in diameter) containing c. 2,500 cells that eventually differentiate and segregate into a large number of daughter colonies that are subsequently liberated. Alternatively, colonies may differentiate into a mass of zoospores that escape and settle to develop into new colonies. In Gl. psammophila, cubic packets of cells form large sticky clusters that bind sand together, while Ga. australica and A. geitleri are unicellular species. Using fixation by high-pressure freezing, a distinctive perforated theca was observed by TEM in all genera of this lineage, and we hypothesize this unique covering may be the first morphological feature to characterize most, if not all, pelagophytes. This study substantiates the diverse nature of sand-dwelling pelagophytes as well as their mechanisms for thriving in a dynamic habitat.

Pleistocene Ice Ages Created New Evolutionary Lineages, but Limited Speciation in Northeast Pacific Winged Kelp.

The extent that Pleistocene climate variability promoted speciation has been much debated. Here, we surveyed genetic markers in winged kelp Alaria in the Gulf of Alaska, Northeast Pacific Ocean to understand how paleoclimates may have influenced diversity in this kelp. The study included wide geographic sampling over 2800 km and large sample sizes compared to previous studies of this kelp. Mitochondrial 5'-COI (664 bp), plastid rbcL-3' (740 bp) and 8 microsatellite markers in 16 populations resolved 5 well-defined lineages. COI-rbcL haplotypes were distributed chaotically among populations around the Gulf of Alaska. Principal Coordinates Analysis of microsatellite genotypes grouped plants largely by organellar lineage instead of geography, indicating reproductive isolation among lineages. However, microsatellite markers detected hybrids at 3 sites where lineages co-occurred. Local adaptation on various time scales may be responsible for some genetic differences between populations located along wave-energy and salinity gradients, but the chaotic pattern of variability over hundreds of kilometers is likely due to isolations in northern refugia during Pleistocene ice ages. The range of divergences between populations indicates that episodic glaciations led to the creation of new lineages, but population turnover (local extinctions and recolonizations) limited the formation of new species in the Northeastern Pacific Ocean.

Unique biodiversity in Arctic marine forests is shaped by diverse recolonization pathways and far northern glacial refugia.

The Arctic is experiencing a rapid shift toward warmer regimes, calling for a need to understand levels of biodiversity and ecosystem responses to climate cycles. This study presents genetic data for 109 Arctic marine forest species (seaweeds), which revealed contiguous populations extending from the Bering Sea to the northwest Atlantic, with high levels of genetic diversity in the east Canadian Arctic. One-fifth of the species sampled appeared restricted to Arctic waters. Further supported by hindcasted species distributions during the Last Glacial Maximum, we hypothesize that Arctic coastal systems were recolonized from many geographically disparate refugia leading to enriched diversity levels in the east Canadian Arctic, with important contributions stemming from northerly refugia likely centered along southern Greenland. Our results suggest Arctic marine biomes persisted through cycles of glaciation, leading to unique assemblages in polar waters, rather than being entirely derived from southerly (temperate) areas following glaciation. As such, Arctic marine species are potentially born from selective pressures during Cenozoic global cooling and eventual ice conditions beginning in the Pleistocene. Arctic endemic diversity was likely additionally driven by repeated isolations into globally disparate refugia during glaciation. This study highlights the need to take stock of unique Arctic marine biodiversity. Amplification of warming and loss of perennial ice cover are set to dramatically alter available Arctic coastal habitat, with the potential loss of diversity and decline in ecosystem resilience.

The importance of taxonomic resolution for additive beta diversity as revealed through DNA barcoding.

Additive diversity partitioning (α, ß, and γ) is commonly used to study the distribution of species-level diversity across spatial scales. Here, we first investigate whether published studies of additive diversity partitioning show signs of difficulty attaining species-level resolution due to inherent limitations with morphological identifications. Second, we present a DNA barcoding approach to delineate specimens of stream caddisfly larvae (order Trichoptera) and consider the importance of taxonomic resolution on classical (additive) measures of beta (ß) diversity. Caddisfly larvae were sampled using a hierarchical spatial design in two regions (subarctic Churchill, Manitoba, Canada; temperate Pennsylvania, USA) and then additively partitioned according to Barcode Index Numbers (molecular clusters that serve as a proxy for species), genus, and family levels; diversity components were expressed as proportional species turnover. We screened 114 articles of additive diversity partitioning and found that a third reported difficulties with achieving species-level identifications, with a clear taxonomic tendency towards challenges identifying invertebrate taxa. Regarding our own study, caddisfly BINs appeared to show greater subregional turnover (e.g., proportional additive ß) compared to genus or family levels. Diversity component studies failing to achieve species resolution due to morphological identifications may therefore be underestimating diversity turnover at larger spatial scales.

Spatial variation in population structure and its relation to movement and the potential for dispersal in a model intertidal invertebrate.

Dispersal, the movement of an individual away from its natal or breeding ground, has been studied extensively in birds and mammals to understand the costs and benefits of movement behavior. Whether or not invertebrates disperse in response to such attributes as habitat quality or density of conspecifics remains uncertain, due in part to the difficulties in marking and recapturing invertebrates. In the upper Bay of Fundy, Canada, the intertidal amphipod Corophium volutator swims at night around the new or full moon. Furthermore, this species is regionally widespread across a large spatial scale with site-to-site variation in population structure. Such variation provides a backdrop against which biological determinants of dispersal can be investigated. We conducted a large-scale study at nine mudflats, and used swimmer density, sampled using stationary plankton nets, as a proxy for dispersing individuals. We also sampled mud residents using sediment cores over 3 sampling rounds (20-28 June, 10-17 July, 2-11 August 2010). Density of swimmers was most variable at the largest spatial scales, indicating important population-level variation. The smallest juveniles and large juveniles or small adults (particularly females) were consistently overrepresented as swimmers. Small juveniles swam at most times and locations, whereas swimming of young females decreased with increasing mud presence of young males, and swimming of large juveniles decreased with increasing mud presence of adults. Swimming in most stages increased with density of mud residents; however, proportionally less swimming occurred as total mud resident density increased. We suggest small juveniles move in search of C. volutator aggregations which possibly act as a proxy for better habitat. We also suggest large juveniles and small adults move if potential mates are limiting. Future studies can use sampling designs over large spatial scales with varying population structure to help understand the behavioral ecology of movement, and dispersal in invertebrate taxa.