Vanessa R. Marcelino

Multi-marker metabarcoding of coral skeletons reveals a rich microbiome and diverse evolutionary origins of endolithic algae.

Bacteria, fungi and green algae are common inhabitants of coral skeletons. Their diversity is poorly characterized because they are difficult to identify with microscopy or environmental sequencing, as common metabarcoding markers have low phylogenetic resolution and miss a large portion of the biodiversity. We used a cost-effective protocol and a combination of markers (tufA, 16S rDNA, 18S rDNA and 23S rDNA) to characterize the microbiome of 132 coral skeleton samples. We identified a wide range of prokaryotic and eukaryotic organisms, many never reported in corals before. We additionally investigated the phylogenetic diversity of the green algae—the most abundant eukaryotic member of this community, for which previous literature recognizes only a handful of endolithic species. We found more than 120 taxonomic units (near species level), including six family-level lineages mostly new to science. The results suggest that the existence of lineages with an endolithic lifestyle predates the existence of modern scleractinian corals by ca. 250my, and that this particular niche was independently invaded by over 20 lineages in green algae evolution. These results highlight the potential of the multi-marker approach to assist in species discovery and, when combined with a phylogenetic framework, clarify the evolutionary origins of host-microbiota associations.

Evolutionary Dynamics of Chloroplast Genomes in Low Light: A Case Study of the Endolithic Green Alga Ostreobium quekettii

Abstract Some photosynthetic organisms live in extremely low light environments. Light limitation is associated with selective forces as well as reduced exposure to mutagens, and over evolutionary timescales it can leave a footprint on species’ genomes. Here, we present the chloroplast genomes of four green algae (Bryopsidales, Ulvophyceae), including the endolithic (limestone-boring) alga Ostreobium quekettii, which is a low light specialist. We use phylogenetic models and comparative genomic tools to investigate whether the chloroplast genome of Ostreobium corresponds to our expectations of how low light would affect genome evolution. Ostreobium has the smallest and most gene-dense chloroplast genome among Ulvophyceae reported to date, matching our expectation that light limitation would impose resource constraints reflected in the chloroplast genome architecture. Rates of molecular evolution are significantly slower along the phylogenetic branch leading to Ostreobium, in agreement with the expected effects of low light and energy levels on molecular evolution. We expected the ability of Ostreobium to perform photosynthesis in very low light to be associated with positive selection in genes related to the photosynthetic machinery, but instead, we observed that these genes may be under stronger purifying selection. Besides shedding light on the genome dynamics associated with a low light lifestyle, this study helps to resolve the role of environmental factors in shaping the diversity of genome architectures observed in nature.

Ecological niche models of invasive seaweeds

Ecological niche models (ENMs) are commonly used to calculate habitat suitability from species’ occurrence and macroecological data. In invasive species biology, ENMs can be applied to anticipate whether invasive species are likely to establish in an area, to identify critical routes and arrival points, to build risk maps and to predict the extent of potential spread following an introduction. Most studies using ENMs focus on terrestrial organisms and applications in the marine realm are still relatively rare. Here, we review some common methods to build ENMs and their application in seaweed invasion biology. We summarize methods and concepts involved in the development of niche models, show examples of how they have been applied in studies on algae and discuss the application of ENMs in invasive algae research and to predict effects of climate change on seaweed distributions.

Taxonomic revision of Halimeda (Bryopsidales, Chlorophyta) in south-western Australia

Abstract. Halimeda J.V.Lamour. is a green algal genus that is an important component of tropical reefs and lagoons. Although it does not generally occur outside the tropics, the range of one putatively widespread species, Halimeda cuneata Hering, in Western Australia extends southward past Cape Leeuwin to its southern extreme at Cape Howe and around to its most easterly record at Middle Island of the Recherche Archipelago. Previous molecular studies have shown that H. cuneata as recorded worldwide encompasses cryptic species diversity, with most of the cryptic entities being geographically isolated from the others. Halimeda cuneata has been the name consistently applied to specimens from the south-western coast of Australia, where it has been regarded as the only representative of its genus in the region. Using a combination of morphological features and assessment of species boundaries based on a plastid gene (tufA) and a nuclear protein-coding gene (HSP90), we have reassessed the supposed presence of H. cuneata in south-western Australia. Our results showed the existence of two species in the region, namely, H. versatilis J.Agardh, to which the name H. cuneata has been misapplied, and H. gigas W.R.Taylor, a central-Pacific species previously recorded only from tropical Australia.

Phylogenetic position of the coral symbiont Ostreobium (Ulvophyceae) inferred from chloroplast genome data

The green algal genus Ostreobium is an important symbiont of corals, playing roles in reef decalcification and providing photosynthates to the coral during bleaching events. A chloroplast genome of a cultured strain of Ostreobium was available, but low taxon sampling and Ostreobiums early‐branching nature left doubt about its phylogenetic position. Here, we generate and describe chloroplast genomes from four Ostreobium strains as well as Avrainvillea mazei and Neomeris sp., strategically sampled early‐branching lineages in the Bryopsidales and Dasycladales respectively. At 80,584 bp, the chloroplast genome of Ostreobium sp. HV05042 is the most compact yet found in the Ulvophyceae. The Avrainvillea chloroplast genome is ~94 kbp and contains introns in infA and cysT that have nearly complete sequence identity except for an open reading frame (ORF) in infA that is not present in cysT. In line with other bryopsidalean species, it also contains regions with possibly bacteria‐derived ORFs. The Neomeris data did not assemble into a canonical circular chloroplast genome but a large number of contigs containing fragments of chloroplast genes and showing evidence of long introns and intergenic regions, and the Neomeris chloroplast genome size was estimated to exceed 1.87 Mb. Chloroplast phylogenomics and 18S nrDNA data showed strong support for the Ostreobium lineage being sister to the remaining Bryopsidales. There were differences in branch support when outgroups were varied, but the overall support for the placement of Ostreobium was strong. These results permitted us to validate two suborders and introduce a third, the Ostreobineae.

Evolutionary dynamics of algal traits and diversity

Algae have a particularly rich evolutionary history that has not yet been comprehensively explored. We review statistical techniques to infer patterns of trait evolution and species diversification from phylogenies. We illustrate these methods using the evolution of algal thermal niches and its interaction with species diversification as a case study. We offer some perspectives for the application of these methods in other fields of phycology and the integration of microand macroevolutionary approaches.

Large Diversity of Nonstandard Genes and Dynamic Evolution of Chloroplast Genomes in Siphonous Green Algae (Bryopsidales, Chlorophyta)

Abstract Chloroplast genomes have undergone tremendous alterations through the evolutionary history of the green algae (Chloroplastida). This study focuses on the evolution of chloroplast genomes in the siphonous green algae (order Bryopsidales). We present five new chloroplast genomes, which along with existing sequences, yield a data set representing all but one families of the order. Using comparative phylogenetic methods, we investigated the evolutionary dynamics of genomic features in the order. Our results show extensive variation in chloroplast genome architecture and intron content. Variation in genome size is accounted for by the amount of intergenic space and freestanding open reading frames that do not show significant homology to standard plastid genes. We show the diversity of these nonstandard genes based on their conserved protein domains, which are often associated with mobile functions (reverse transcriptase/intron maturase, integrases, phage- or plasmid-DNA primases, transposases, integrases, ligases). Investigation of the introns showed proliferation of group II introns in the early evolution of the order and their subsequent loss in the core Halimedineae, possibly through RT-mediated intron loss.

High-throughput sequencing for algal systematics

ABSTRACT In recent years, the use of molecular data in algal systematics has increased as high-throughput sequencing (HTS) has become more accessible, generating very large datasets at a reasonable cost. In this perspectives paper, our goal is to describe how HTS technologies can advance algal systematics. Following an introduction to some common HTS technologies, we discuss how metabarcoding can accelerate algal species discovery. We show how various HTS methods can be applied to generate datasets for accurate species delimitation, and how HTS can be applied to historical type specimens to assist the nomenclature process. Finally, we discuss how HTS data such as organellar genomes and transcriptomes can be used to construct well-resolved phylogenies, leading to a stable and natural classification of algal groups. We include examples of bioinformatic workflows that may be applied to process data for each purpose, along with common programs used to achieve each step. We also discuss possible strategies and the new skill set that will be required to fully embrace HTS as a part of algal systematics, along with considerations of cost and experimental design. HTS technology has revolutionized many fields in biology, and will certainly do the same in algal systematics.

Diversity and stability of coral endolithic microbial communities at a naturally high p CO2 reef

The health and functioning of reef‐building corals is dependent on a balanced association with prokaryotic and eukaryotic microbes. The coral skeleton harbours numerous endolithic microbes, but their diversity, ecological roles and responses to environmental stress, including ocean acidification (OA), are not well characterized. This study tests whether pH affects the diversity and structure of prokaryotic and eukaryotic algal communities associated with skeletons of Porites spp. using targeted amplicon (16S rRNA gene, UPA and tufA) sequencing. We found that the composition of endolithic communities in the massive coral Porites spp. inhabiting a naturally high pCO2 reef (avg. pCO2 811 μatm) is not significantly different from corals inhabiting reference sites (avg. pCO2 357 μatm), suggesting that these microbiomes are less disturbed by OA than previously thought. Possible explanations may be that the endolithic microhabitat is highly homeostatic or that the endolithic micro‐organisms are well adapted to a wide pH range. Some of the microbial taxa identified include nitrogen‐fixing bacteria (Rhizobiales and cyanobacteria), algicidal bacteria in the phylum Bacteroidetes, symbiotic bacteria in the family Endozoicomoniaceae, and endolithic green algae, considered the major microbial agent of reef bioerosion. Additionally, we test whether host species has an effect on the endolithic community structure. We show that the endolithic community of massive Porites spp. is substantially different and more diverse than that found in skeletons of the branching species Seriatopora hystrix and Pocillopora damicornis. This study reveals highly diverse and structured microbial communities in Porites spp. skeletons that are possibly resilient to OA.

Reference datasets of tufA and UPA markers to identify algae in metabarcoding surveys

The data presented here are related to the research article “Multi-marker metabarcoding of coral skeletons reveals a rich microbiome and diverse evolutionary origins of endolithic algae” (Marcelino and Verbruggen, 2016) [1]. Here we provide reference datasets of the elongation factor Tu (tufA) and the Universal Plastid Amplicon (UPA) markers in a format that is ready-to-use in the QIIME pipeline (Caporaso et al., 2010) [2]. In addition to sequences previously available in GenBank, we included newly discovered endolithic algae lineages using both amplicon sequencing (Marcelino and Verbruggen, 2016) [1] and chloroplast genome data (Marcelino et al., 2016; Verbruggen et al., in press) [3], [4]. We also provide a script to convert GenBank flatfiles into reference datasets that can be used with other markers. The tufA and UPA reference datasets are made publicly available here to facilitate biodiversity assessments of microalgal communities.