A new centrohelid heliozoan, Pterocystis polycristalepis sp. nov., and taxonomic and phylogenetic concerns within Pterista (Haptista: Centroplasthelida).

A new species of centrohelid heliozoans, Pterocystis polycristalepis sp. nov. (Pterocystidae), was examined using light and electron microscopy. The novel centrohelid is characterized by the presence of leaf-like spine-scales with a broad pedicel-like structure on the proximal part and many subparallel ribs on the lateral wing surface. The plate-scales are ovoid with medial tubular thickening and many subparallel ribs on the very extensive marginal rim. The closely related species Pterocystis striata has also been studied in detail using light and electron microscopy. Phylogenetic analysis of 18S rRNA gene sequences placed both species into a separate clade within Pterista. The closest morphologically characterized species to the new clade is Triangulopteris lacunata. The 18S rRNA sequence of Pseudoraphidiophrys veliformis was grouped within Pterista and found to be closely related to Pterocystis polycristalepis, Pterocystis striata, and Triangulopteris lacunata. Cyst-scales of various shapes, cell and cyst aggregations, syncytia, and a cell with a stalk were revealed in a clonal culture of P. veliformis. Analysis of the morphology and phylogenetic position of the studied species and other centrohelids revealed a large number of taxonomic and phylogenetic problems in Pterista.

Complex morphologies of biogenic crystals emerge from anisotropic growth of symmetry-related facets.

Directing crystal growth into complex morphologies is challenging, as crystals tend to adopt thermodynamically stable morphologies. However, many organisms form crystals with intricate morphologies, as exemplified by coccoliths, microscopic calcite crystal arrays produced by unicellular algae. The complex morphologies of the coccolith crystals were hypothesized to materialize from numerous crystallographic facets, stabilized by fine-tuned interactions between organic molecules and the growing crystals. Using electron tomography, we examined multiple stages of coccolith development in three dimensions. We found that the crystals express only one set of symmetry-related crystallographic facets, which grow differentially to yield highly anisotropic shapes. Morphological chirality arises from positioning the crystals along specific edges of these same facets. Our findings suggest that growth rate manipulations are sufficient to yield complex crystalline morphologies.

Morphological development of Pleurochrysis carterae coccoliths examined by cryo-electron tomography.

Coccolithophores are single-celled marine algae that produce calcified scales called coccoliths. Each scale is composed of anvil-shaped single crystals of calcite that are mechanically interlocked, constituting a remarkable example of the multi-level construction of mineralized structures. Coccolith formation starts with the nucleation of rhombohedral crystals on an organic substrate called base plate. The crystals then grow preferentially along specific directions to generate the mature structure, which is then transported to the outside of the cells. Here, we extracted forming coccoliths from Pleurochrysis carterae cells and used cryo-electron tomography to characterize, in their native, hydrated state, the three-dimensional morphology and arrangement of the crystals. Comparing the crystal morphology across three different stages of coccolith formation, we show that competition for space between adjacent crystals contributes significantly to regulation of morphology by constraining growth in certain directions. We further demonstrate that crystals within a coccolith ring develop at different rates and that each crystalline unit rests directly in contact with the base plate and overgrow the rim of the organic substrate during development.

Three-dimensional architecture and surface functionality of coccolith base plates.

Coccolithophores are marine phytoplankton that are among the most prolific calcifiers widespread in Earth's oceans, playing a crucial role in the carbon cycle and in the transport of organic matter to the deep sea. These organisms produce highly complex mineralized scales that are composed of hierarchical assemblies of nano-crystals of calcium carbonate in the form of calcite. Coccolith formation in vivo occurs within compartmentalized mineralisation vesicles derived from the Golgi body, which contain coccolith-associated polysaccharides ('CAPs') providing polymorph selection and mediating crystal growth kinetics, and oval organic mineralisation templates, also known as base plates, which promote heterogenous nucleation and further mechanical interlocking of calcite single crystals. Although the function of coccolith base plates in controlling crystal nucleation have been widely studied, their 3D spatial organization and the chemical functional groups present on the crystal nucleation sites, which are two crucial features impacting biomineralization, remain unsolved. Utilising cryo-electron tomography we show that base plates derived from an exemplary coccolithophore Pleurochrysis carterae (Pcar) in their native hydrated state have a complex 3-layered structure. We further demonstrate, for the first time, the edge and rim of the base plate - where the crystals nucleate - are rich in primary amine functionalities that provide binding targets for negatively charged complexes composed of synthetic macromolecules and Ca2+ ions. Our results indicate that electrostatic interactions between the negatively charged biogenic CAPs and the positively charged rim of the base plate are sufficient to mediate the transport of Ca2+ cations to the mineralization sites.

Emiliania huxleyi coccolith calcite mass modulation by morphological changes and ecology in the Mediterranean Sea.

To understand the response of marine calcifying organisms under high CO2 scenarios, it is critical to study their calcification patterns in the natural environment. This paper focuses on a major calcifying phytoplankton group, the coccolithophores, through the analysis of water samples collected along a W-E Mediterranean transect during two research cruises, in April 2011 (Meteor cruise M84/3) and May 2013 (MedSeA cruise 2013). The Mediterranean Sea is a marginal sea characterized by large biogeochemical gradients. Currently, it is undergoing both warming and ocean acidification, processes which are rapidly modifying species distribution and calcification. The species Emiliania huxleyi largely dominates the total coccolithophore production in present day oceans and marine basins, including the Mediterranean Sea. A series of morphometric measurements were performed on the coccoliths of this species to estimate their mass, length and calculate a calcification index (proxy for the size-normalized calcification degree). The most abundant morphotype of E. huxleyi in the Mediterranean Sea is Type A. Coccoliths of this morphotype were additionally analyzed based on scanning electron microscopy images: four calcification varieties were quantified, according to the relationship between slit length-tube width, and the state of the central area (open or closed). The average E. huxleyi coccolith mass along the Mediterranean oceanographic transect depended strongly on both the average coccolith length and calcification index. The variability in average coccolith length and calcification index across samples reflected oscillations in the relative abundance of the calcification varieties. We also demonstrated that the distribution of the calcification varieties followed the main environmental gradients (carbonate chemistry, salinity, temperature, nutrient concentrations). Hence, shifts in the distribution of the calcification varieties and of the average E. huxleyi coccolith mass are to be expected in the Mediterranean Sea under climate change. These physiological and ecological responses will modulate the net coccolithophore calcification and, ultimately, the regional carbonate export to the seafloor.

The requirement for calcification differs between ecologically important coccolithophore species.

Coccolithophores are globally distributed unicellular marine algae that are characterized by their covering of calcite coccoliths. Calcification by coccolithophores contributes significantly to global biogeochemical cycles. However, the physiological requirement for calcification remains poorly understood as non-calcifying strains of some commonly used model species, such as Emiliania huxleyi, grow normally in laboratory culture. To determine whether the requirement for calcification differs between coccolithophore species, we utilized multiple independent methodologies to disrupt calcification in two important species of coccolithophore: E. huxleyi and Coccolithus braarudii. We investigated their physiological response and used time-lapse imaging to visualize the processes of calcification and cell division in individual cells. Disruption of calcification resulted in major growth defects in C. braarudii, but not in E. huxleyi. We found no evidence that calcification supports photosynthesis in C. braarudii, but showed that an inability to maintain an intact coccosphere results in cell cycle arrest. We found that C. braarudii is very different from E. huxleyi as it exhibits an obligate requirement for calcification. The identification of a growth defect in C. braarudii resulting from disruption of the coccosphere may be important in considering their response to future changes in ocean carbonate chemistry.

Virus-induced apoptosis and phosphorylation form of metacaspase in the marine coccolithophorid Emiliania huxleyi.

Lytic viral infection and programmed cell death (PCD) are thought to represent two distinct death mechanisms in phytoplankton, unicellular photoautotrophs that drift with ocean currents. PCD (apoptosis) is mainly brought about by the activation of caspases, a protease family with unique substrate selectivity. Here, we demonstrated that virus infection induced apoptosis of marine coccolithophorid Emiliania huxleyi BOF92 involving activation of metacaspase. E. huxleyi cells exhibited cell death process akin to that of apoptosis when exposed to virus infection. We observed typical hallmarks of apoptosis including cell shrinkage, associated nuclear morphological changes and DNA fragmentation. Immunoblotting revealed that antibody against human active-caspase-3 shared epitopes with a protein of ≈ 23 kDa; whose pattern of expression correlated with the onset of cell death. Moreover, analysis on two-dimensional gel electrophoresis revealed that two spots of active caspase-3 co-migrated with the different isoelectric points. Phosphatase treatment of cytosolic extracts containing active caspases-3 showed a mobility shift, suggesting that phosphorylated form of this enzyme might be present in the extracts. Computational prediction of phosphorylation sites based on the amino acid sequence of E. huxleyi metacaspase showed multiple phosphorylated sites for serine, threonine and tyrosine residues. This is the first report showing that phosphorylation modification of metacaspase in E. huxleyi might be required for certain biochemical and morphological changes during virus induced apoptosis.

Extreme strontium concentrations reveal specific biomineralization pathways in certain coccolithophores with implications for the Sr/Ca paleoproductivity proxy.

The formation of the coccolith biominerals by a group of marine algae (the Coccolithophores) offers fascinating research avenues both from the biological and geological sides. It is surprising how biomineralisation by a key phytoplanktonic group remains underconstrained, yet is influential on ocean alkalinity and responsible for the built up of our paleoclimatic archive over the last 200 Myrs. Here, we report two close relative coccolith taxa exhibiting substantial bioaccumulation of strontium: Scyphosphaera and Pontosphaera grown in the laboratory or retrieved from Pliocene sediments. This strontium enrichment relative to calcium is one order of magnitude greater than reported in other coccoliths of the orders Isochrysidales and Coccolithales, and extends well beyond established controls on Sr/Ca ratios by temperature and growth rate. We discuss this prominent vital effect in relation with possible specific uptake of strontium relative to calcium from the extracellular environment to the coccolith vesicle in coccolithophores excreting very large scale coccoliths. The report of Sr-rich biominerals challenges our current understanding of the cellular acquisition and intracellular trafficking of alkaline earth cations in phytoplanktonic calcifying eukaryotic algae. The presence of Sr-rich coccolith species in the geological record has to be quantitatively considered in future Sr/Ca-based palaeoceanographic reconstruction.

Haptophyte Diversity and Vertical Distribution Explored by 18S and 28S Ribosomal RNA Gene Metabarcoding and Scanning Electron Microscopy.

Haptophyta encompasses more than 300 species of mostly marine pico- and nanoplanktonic flagellates. Our aims were to investigate the Oslofjorden haptophyte diversity and vertical distribution by metabarcoding, and to improve the approach to study haptophyte community composition, richness and proportional abundance by comparing two rRNA markers and scanning electron microscopy (SEM). Samples were collected in August 2013 at the Outer Oslofjorden, Norway. Total RNA/cDNA was amplified by haptophyte-specific primers targeting the V4 region of the 18S, and the D1-D2 region of the 28S rRNA. Taxonomy was assigned using curated haptophyte reference databases and phylogenetic analyses. Both marker genes showed Chrysochromulinaceae and Prymnesiaceae to be the families with highest number of Operational Taxonomic Units (OTUs), as well as proportional abundance. The 18S rRNA data set also contained OTUs assigned to eight supported and defined clades consisting of environmental sequences only, possibly representing novel lineages from family to class. We also recorded new species for the area. Comparing coccolithophores by SEM with metabarcoding shows a good correspondence with the 18S rRNA gene proportional abundances. Our results contribute to link morphological and molecular data and 28S to 18S rRNA gene sequences of haptophytes without cultured representatives, and to improve metabarcoding methodology.

Coccospheres confer mechanical protection: New evidence for an old hypothesis.

UNLABELLED: Emiliania huxleyi has evolved an extremely intricate coccosphere architecture. The coccosphere is comprised of interlocking coccoliths embedded in a polysaccharide matrix. In this work, we performed in-situ scanning electron microscopy based compression tests and conclude that coccospheres have a mechanical protection function. The coccosphere exhibits exceptional damage tolerance in terms of inelastic deformation, recovery and stable crack growth before catastrophic fracture, a feature, which is not found in monolithic ceramic structures. Some of the mechanical features of the coccospheres are due to their architecture, especially polysaccharide matrix that acts as a kind of bio-adhesive. Our data provide strong evidence for the mechanical protection-hypothesis of coccolithophore calcification, without excluding other functions of calcification such as various biochemical roles discussed in the literature. STATEMENT OF SIGNIFICANCE: Although bio-mechanics of shell structures like nacre have been studied over the past decade, coccospheres present an architecture that is quite distinct and complex. It is a porous cell structure evolved to protect the living algae cell inside it in the oceans, subjected to significant hydrostatic pressure. Despite being made of extremely brittle constituents like calcium carbonate, our study finds that coccospheres possess significant damage tolerance especially due to their interlocking coccolith architecture. This will have consequences in bio-mimetic design, especially relating to high pressure applications.

Virocell Metabolism: Metabolic Innovations During Host-Virus Interactions in the Ocean.

Marine viruses are considered to be major ecological, evolutionary, and biogeochemical drivers of the marine environment, responsible for nutrient recycling and determining species composition. Viruses can re-shape their host's metabolic network during infection, generating the virocell-a unique metabolic state that supports their specific requirement. Here we discuss the concept of 'virocell metabolism' and its formation by rewiring of host-encoded metabolic networks, or by introducing virus-encoded auxiliary metabolic genes which provide the virocell with novel metabolic capabilities. The ecological role of marine viruses is commonly assessed by their relative abundance and phylogenetic diversity, lacking the ability to assess the dynamics of active viral infection. The new ability to define a unique metabolic state of the virocell will expand the current virion-centric approaches in order to quantify the impact of marine viruses on microbial food webs.

A vacuole-like compartment concentrates a disordered calcium phase in a key coccolithophorid alga.

Coccoliths are calcitic particles produced inside the cells of unicellular marine algae known as coccolithophores. They are abundant components of sea-floor carbonates, and the stoichiometry of calcium to other elements in fossil coccoliths is widely used to infer past environmental conditions. Here we study cryo-preserved cells of the dominant coccolithophore Emiliania huxleyi using state-of-the-art nanoscale imaging and spectroscopy. We identify a compartment, distinct from the coccolith-producing compartment, filled with high concentrations of a disordered form of calcium. Co-localized with calcium are high concentrations of phosphorus and minor concentrations of other cations. The amounts of calcium stored in this reservoir seem to be dynamic and at a certain stage the compartment is in direct contact with the coccolith-producing vesicle, suggesting an active role in coccolith formation. Our findings provide insights into calcium accumulation in this important calcifying organism.

Phosphorus starvation induces membrane remodeling and recycling in Emiliania huxleyi.

Nutrient availability is an important factor controlling phytoplankton productivity. Phytoplankton contribute c. 50% of the global photosynthesis and possess efficient acclimation mechanisms to cope with nutrient stress. We investigate the cellular response of the bloom-forming coccolithophore Emiliania huxleyi to phosphorus (P) scarcity, which is often a limiting factor in marine ecosystems. We combined mass spectrometry, fluorescence microscopy, transmission electron microscopy (TEM) and gene expression analyses in order to assess diverse cellular features in cells exposed to P limitation and recovery. Early starvation-induced substitution of phospholipids in the cells' membranes with galacto- and betaine lipids. Lipid remodeling was rapid and reversible upon P resupply. The PI3K inhibitor wortmannin reduced phospholipid substitution, suggesting a possible involvement of PI3K- signaling in this process. In addition, P limitation enhanced the formation and acidification of membrane vesicles in the cytoplasm. Intracellular vesicles may facilitate the recycling of cytoplasmic content, which is engulfed in the vesicles and delivered to the main vacuole. Long-term starvation was characterized by a profound increase in cell size and morphological alterations in cellular ultrastructure. This study provides cellular and molecular basis for future ecophysiological assessment of natural E. huxleyi populations in oligotrophic regions.

Lysing bloom-causing alga Phaeocystis globosa with microbial algicide: An efficient process that decreases the toxicity of algal exudates.

Algicidal microbes could effectively remove the harmful algae from the waters. In this study, we were concerned with the ecological influence of an algicide extracted from Streptomyces alboflavus RPS, which could completely lyse the Phaeocystis globosa cells within two days. In microcosms, 4 µg/mL of the microbial algicide could efficiently remove P. globosa cells without suppressing other aquatic organisms. Bioluminescent assays confirmed that the toxicity of microbial algicide at this concentration was negligible. Interestingly, the toxicity of P. globosa exudates was also significantly reduced after being treated with the algicide. Further experiments revealed that the microbial algicide could instantly increase the permeability of the plasma membrane and disturb the photosynthetic system, followed by the deformation of organelles, vacuolization and increasing oxidative stress. The pre-incubation of N-acetyl cysteine (NAC) verified that the rapid damages to the plasma membrane and photosynthetic system caused the algal death in the early phase, and the increasing oxidative stress killed the rest. The late accumulation and possible release of CAT also explained the decreasing toxicity of the algal culture. These results indicated that this microbial algicide has great potential in controlling the growth of P. globosa on site.

A role for diatom-like silicon transporters in calcifying coccolithophores.

Biomineralization by marine phytoplankton, such as the silicifying diatoms and calcifying coccolithophores, plays an important role in carbon and nutrient cycling in the oceans. Silicification and calcification are distinct cellular processes with no known common mechanisms. It is thought that coccolithophores are able to outcompete diatoms in Si-depleted waters, which can contribute to the formation of coccolithophore blooms. Here we show that an expanded family of diatom-like silicon transporters (SITs) are present in both silicifying and calcifying haptophyte phytoplankton, including some globally important coccolithophores. Si is required for calcification in these coccolithophores, indicating that Si uptake contributes to the very different forms of biomineralization in diatoms and coccolithophores. Significantly, SITs and the requirement for Si are absent from highly abundant bloom-forming coccolithophores, such as Emiliania huxleyi. These very different requirements for Si in coccolithophores are likely to have major influence on their competitive interactions with diatoms and other siliceous phytoplankton.

Incorporation of zinc into the coccoliths of the microalga Emiliania huxleyi.

The coccolithophore Emiliania huxleyi is covered with elaborated calcite plates, the so-called coccoliths, which are produced inside the cells. We investigated the incorporation of zinc into the coccoliths of E. huxleyi by applying different zinc and calcium amounts via the culture media and subsequently analyzing the zinc content in the cells and the Zn/Ca ratio of the coccoliths. To investigate the Zn/Ca ratio of coccoliths built in the manipulated media, the algae have first to be decalcified, i.e. coccolith free. We used a newly developed decalcification method to obtain 'naked' cells for cultivation. E. huxleyi proliferated and produced new coccoliths in all media with manipulated Zn/Ca ratios. The cells and the newly built coccoliths were investigated regarding their zinc content and their Zn/Ca ratio, respectively. High zinc amounts were taken up by the algae. The Zn/Ca ratio of the coccoliths was positively correlated to the Zn/Ca ratio of the applied media. The unique feature of the coccoliths was maintained also at high Zn/Ca ratios. We suggest the following pathway of the zinc ions into the coccoliths: first, the zinc ions are bound to the cell surface, followed by their transportation into the cytoplasm. Obviously, the zinc ions are removed afterwards into the coccolith vesicle, where the zinc is incorporated into the calcite coccoliths which are then extruded. The incorporation of toxic zinc ions into the coccoliths possibly due to a new function of the coccoliths as detoxification sites is discussed.

Decrease in coccolithophore calcification and CO2 since the middle Miocene.

Marine algae are instrumental in carbon cycling and atmospheric carbon dioxide (CO2) regulation. One group, coccolithophores, uses carbon to photosynthesize and to calcify, covering their cells with chalk platelets (coccoliths). How ocean acidification influences coccolithophore calcification is strongly debated, and the effects of carbonate chemistry changes in the geological past are poorly understood. This paper relates degree of coccolith calcification to cellular calcification, and presents the first records of size-normalized coccolith thickness spanning the last 14 Myr from tropical oceans. Degree of calcification was highest in the low-pH, high-CO2 Miocene ocean, but decreased significantly between 6 and 4 Myr ago. Based on this and concurrent trends in a new alkenone ɛp record, we propose that decreasing CO2 partly drove the observed trend via reduced cellular bicarbonate allocation to calcification. This trend reversed in the late Pleistocene despite low CO2, suggesting an additional regulator of calcification such as alkalinity.

Coccolithophore biomineralization: New questions, new answers.

Coccolithophores are unicellular phytoplankton that are characterized by the presence intricately formed calcite scales (coccoliths) on their surfaces. In most cases coccolith formation is an entirely intracellular process - crystal growth is confined within a Golgi-derived vesicle. A wide range of coccolith morphologies can be found amongst the different coccolithophore groups. This review discusses the cellular factors that regulate coccolith production, from the roles of organic components, endomembrane organization and cytoskeleton to the mechanisms of delivery of substrates to the calcifying compartment. New findings are also providing important information on how the delivery of substrates to the calcification site is co-ordinated with the removal of H(+) that are a bi-product of the calcification reaction. While there appear to be a number of species-specific features of the structural and biochemical components underlying coccolith formation, the fluxes of Ca(2+) and a HCO3(-) required to support coccolith formation appear to involve spatially organized recruitment of conserved transport processes.

Bio-optic characterization of Discosphaera tubifer bloom occurs in an overcrowded fishing harbour at Veraval, India.

Discosphaera tubifer, a coccolithophore has been first time reported as a bloom-forming organism from an over-crowded fishing harbour at Veraval, west coast of India. Physiochemical and optical parameters were measured following standard protocols. Average concentration of inorganic nutrients, such as NO2-N (17.26 ± 2.92 µM), NO3-N (643.80 ± 215.99 µM), PO4-P (74.10 ± 26.52 µM) and SiO3-Si (137.66 ± 25.83 µM) were recorded as very high at Veraval port as compared to other coastal stations i.e., 1.48 ± 0.66, 49.16 ± 13.73, 10.03 ± 5.31 and 96.23 ± 22.74 µM, respectively. The pH and salinity (‰) were observed to be low (7.80 ± 0.15 and 28.00 ± 4.54 ‰) as compared to coastal seawaters (8.34 ± 0.06 and 33.24 ± 2.32 ‰). Scanning electron microscopy (SEM) and spectral signature (absorbance and reflectance) study revealed that the bloom-forming organism was D. tubifer. High-performance liquid chromatography (HPLC) study detected that chlorophyllide-a represent nearly 47.53 % of total pigment composition followed by chlorophyll c2 (27.40 %) and chlorophyll c3 (14.25 %). Four prominent absorption peaks were observed within 350 to 700 nm. The first peak was very wide and ranged from 350 to 530 nm and the rest of the three peaks ranged from 550 to 590, 590 to 650 and 650 to 690 nm, respectively. In case of reflection, three peaks appeared between 550 and 590, 590 and 630 and 630 and 670 nm. Nearly 100 % reflection was observed after 720 nm. The eutrophic condition of the port water along with low salinity and low pH might be the reason for D. tubifer bloom formation. This species-specific spectral signature of the D. tubifer bloom may be helpful for developing algorithm of remote sensing data analysis.

Morphological and Phylogenetic Characterization of New Gephyrocapsa Isolates Suggests Introgressive Hybridization in the Emiliania/Gephyrocapsa Complex (Haptophyta).

The coccolithophore genus Gephyrocapsa contains a cosmopolitan assemblage of pelagic species, including the bloom-forming Gephyrocapsa oceanica, and is closely related to the emblematic coccolithophore Emiliania huxleyi within the Noëlaerhabdaceae. These two species have been extensively studied and are well represented in culture collections, whereas cultures of other species of this family are lacking. We report on three new strains of Gephyrocapsa isolated into culture from samples from the Chilean coastal upwelling zone using a novel flow cytometric single-cell sorting technique. The strains were characterized by morphological analysis using scanning electron microscopy and phylogenetic analysis of 6 genes (nuclear 18S and 28S rDNA, plastidial 16S and tufA, and mitochondrial cox1 and cox3 genes). Morphometric features of the coccoliths indicate that these isolates are distinct from G. oceanica and best correspond to G. muellerae. Surprisingly, both plastidial and mitochondrial gene phylogenies placed these strains within the E. huxleyi clade and well separated from G. oceanica isolates, making Emiliania appear polyphyletic. The only nuclear sequence difference, 1bp in the 28S rDNA region, also grouped E. huxleyi with the new Gephyrocapsa isolates and apart from G. oceanica. Specifically, the G. muellerae morphotype strains clustered with the mitochondrial ß clade of E. huxleyi, which, like G. muellerae, has been associated with cold (temperate and sub-polar) waters. Among putative evolutionary scenarios that could explain these results we discuss the possibility that E. huxleyi is not a valid taxonomic unit, or, alternatively the possibility of past hybridization and introgression between each E. huxleyi clade and older Gephyrocapsa clades. In either case, the results support the transfer of Emiliania to Gephyrocapsa. These results have important implications for relating morphological species concepts to ecological and evolutionary units of diversity.