A molecular survey of Ulva sensu lato on German Baltic- and North Sea coasts: A revision of the species inventory reveals their distribution and contributes new taxonomic insights

In this doctoral project, I investigated the recent inventory, distribution and phylogenetic relationships of Ulva sensu lato in northern Germany, including sampling sites at the Baltic Sea, Wadden Sea and on Helgoland. Furthermore, I compared the recent results with historic findings. Therfore, this thesis constitutes a complete revision of the species inventory of Ulva sensu lato in northern Germany. Assessments of biodiversity were based on both the analysis of classical morphological characters and DNA barcoding. Phylogenetic analysis of more than 370 sequences of the tufA marker gene revealed the presence of 20 different species in German waters

DNA barcoding of the German green supralittoral zone indicates the distribution and phenotypic plasticity of Blidingia species and reveals Blidingia cornuta sp. nov.

In temperate and subarctic regions of the Northern Hemisphere, green algae of the genus Blidingia are a substantial and environment‐shaping component of the upper and mid‐supralittoral zones. However, taxonomic knowledge on these important green algae is still sparse. In the present study, the molecular diversity and distribution of Blidingia species in the German State of Schleswig‐Holstein was examined for the first time, including Baltic Sea and Wadden Sea coasts and the off‐shore island of Helgoland (Heligoland). In total, three entities were delimited by DNA barcoding, and their respective distributions were verified (in decreasing order of abundance: Blidingia marginata, Blidingia cornuta sp. nov. and Blidingia minima). Our molecular data revealed strong taxonomic discrepancies with historical species concepts, which were mainly based on morphological and ontogenetic characters. Using a combination of molecular, morphological and ontogenetic approaches, we were able to disentangle previous mis‐identifications of B. minima and demonstrate that the distribution of B. minima is more restricted than expected within the examined area. Blidingia minima, the type of the genus name Blidingia, is epitypified within this study by material collected at the type locality Helgoland. In contrast with B. minima, B. marginata shows a higher phenotypic plasticity and is more widely distributed in the study area than previously assumed. The third entity, Blidingia cornuta sp. nov., is clearly delimited from other described Blidingia species, due to unique characters in its ontogenetic development and morphology as well as by its tufA and rbcL sequences

Molekulare Überprüfung von Ulva sensu lato entlang der deutschen Ost- und Nordseeküste: Eine Überarbeitung des Artinventars trägt neue Erkenntnisse zu deren Verbeitung und Taxonomie bei

In this doctoral project, I investigated the recent inventory, distribution and phylogenetic relationships of Ulva sensu lato in northern Germany, including sampling sites at the Baltic Sea, Wadden Sea and on Helgoland. Furthermore, I compared the recent results with historic findings. Therfore, this thesis constitutes a complete revision of the species inventory of Ulva sensu lato in northern Germany. Assessments of biodiversity were based on both the analysis of classical morphological characters and DNA barcoding. Phylogenetic analysis of more than 370 sequences of the tufA marker gene revealed the presence of 20 different species in German waters.In der vorliegenden Doktorarbeit wurde nicht nur das heutige Artinventar von Ulva sensu lato in Deutschland untersucht, sondern ebenfalls die geografische Verbreitung von Arten und deren phylogenetische Zusammenhänge aufgeklärt. Hierfür wurden beide Küstengebiete, Nordsee und Ostsee, sowie die Hochseeinsel Helgoland berücksichtigt. Weiterhin wurden die von mir in dieser Doktorarbeit erhobenen Daten mit vorherigen Untersuchungen und historischem Material verglichen. Somit stellt diese These eine komplette Überarbeitung des Artinventars von Ulva sensu lato in Norddeutschland dar. Erhebungen der Biodiversität basierten auf Analysen klassisch- morphologischer Charakteristika in Kombination mit der Methode des DNA barcodings, welches kurze Genabschnitte vergleichenden Identitätsüberprüfung eines Organismus nutzt. Durch phylogenetische Analysen von mehr als 370 Gensequenzen des tufA-Markergens wurde die Existenz von 20 verschiedenen Arten nachgewiesen

A tufA metabarcoding approach for Ulva and related seaweeds

Ulva-like green algae are notoriously difficult to distinguish due to their morphological variability and/or similarity. DNA barcoding approaches are therefore currently essential for their reliable identification. However, such approaches often fail when rare or inconspicuous species are to be detected in large mixed populations of Ulva species, for example, at early stages following the introduction of species into new habitats. We therefore developed a detection method based on next-generation DNA sequencing. The approach is suitable for the analysis of DNA traces in preserved water samples or in particles enriched by filtration from such samples. A new pair of primers was designed to amplify a 475 bp segment within the tufA marker gene. The primers were relatively group specific. 68.5% of all reads obtained after quality filtering represented the genus Ulva, 11.1% other Ulvophyceae, and only 20% other Chlorophyta, despite their relatively higher abundance in phytoplankton. The relatively short target amplicon still allows good differentiation of Ulvales and Ulothrichales at the species level. Using a database containing tufA sequences of 879 species - 281 of which were Ulvophyceae and 35 Ulva - we were able to detect mostly Ulvophyceae that had been previously detected in our study area in northern Germany using Sanger sequencing. However, the number of species detected at individual sites was generally higher than in previous studies, which could be due to drifting DNA: Analysis of samples collected at different distances from shore suggests that a sample collected at a given site may be influenced by Ulvophyceae within a radius of up to about 1 km in winter. In summer, this radius is reduced to less than 100 m, possibly due to the less frequent occurrence of strong wind events. Nonetheless, rare species may be detected with this new approach: At one site, an undescribed Blidingia species that was not previously known from our study area was repeatedly detected. Based on these findings, the species was searched for and found, and its identity confirmed by traditional tufA barcoding

Cryptic, alien and lost species: molecular diversity of Ulva sensu lato along the German coasts of the North and Baltic Seas

DNA barcoding analysis, using tufA, revealed considerable differences between the expected and observed species inventory of Ulva sensu lato in the Baltic and North Sea areas of the German state of Schleswig-Holstein. Of 20 observed genetic entities, at least four (U. australis, U. californica, U. gigantea and Umbraulva dangeardii) had been introduced recently, whereas three others (one Ulva sp. and two Blidingia spp.) could not be identified at the species level and could also represent recently introduced species. In addition, the observed distributions of Kornmannia leptoderma and U. rigida were much more extensive than indicated by historical records, whereas Blidingia minima and Gayralia oxysperma were absent or much less common than expected. Barcoding analysis also revealed that both U. tenera (type material) and U. pseudocurvata (historical vouchers) from Helgoland, an off-shore island in the North Sea, actually belong to U. lactuca, a species that appears to be restricted to this island. Furthermore, past morphological descriptions of U. intestinalis and U. compressa have apparently been too restrictive and have been responsible for numerous misidentifications. The same is true for U. linza, which, in northern Germany, clusters into two genetically closely related but morphologically indistinguishable entities. One of these entities is present on Helgoland, while the second is present on North Sea and Baltic Sea mainland coasts

Surveying seaweeds from the Ulvales and Fucales in the world’s most frequently used artificial waterway, the Kiel Canal

The Kiel Canal is one of the world’s most frequently used inland waterways and connects the SW Baltic Sea with the Wadden Sea. At the same time, the canal is a highly eutrophicated environment that is characterized by salinities that range from 3 to 16. This brackish character could make the Kiel Canal an important stepping stone for the introductions of species into the inner Baltic Sea. It could also hinder the identification of native and introduced species, given the fact that salinity sometimes severely affects algal morphology. Here we report on a survey of introduced and native seaweed species in the canal, focusing on the dominant groups, which are Fucales and Ulvales. Of the Fucales, the introduced species Fucus evanescens was detected nearly exclusively inside the canal, while Fucus vesiculosus dominated rockweed communities directly outside the sluice gates. Morphological analysis and genetic barcoding distinguished three species of Ulvales, Ulva linza, Ulva intestinalis and an unknown and possibly introduced species of the genus Blidingia. Species distributions and – in the case of U. intestinalis – branching patterns were clearly affected by salinity, while thallus sizes appeared to be affected by the specific eutrophication status of sites within the canal

Combining pressing and alkaline extraction to increase protein yield from Ulva fenestrata biomass

Many seaweed species have a high production potential and attract interest as future protein sources. A high fiber and ash content, however, demand extraction of the protein to improve its digestibility and protein utilization in food or feed. This study explores three different approaches for protein extraction from Ulva fenestrata in order to maximize the protein extraction yield. Soluble protein was recovered either by mechanical pressing or by homogenization and osmotic shock of the biomass followed by alkaline extraction. The soluble protein was then concentrated by isoelectric precipitation. A combined procedure was carried out by pressing the biomass and following subjecting the residual pulp fraction to homogenization, osmotic shock and alkaline extraction. The three methods were ranked as follows with respect to protein extraction yield (as % of biomass protein); the combined method (23.9 \ub1 0.3%)> the alkaline extraction (6.8 \ub1 0.2%)> mechanical pressing (5.0 \ub1 0.2%). The significant increase when combining the methods was ascribed to a high precipitation yield after alkaline extraction of the pulp, hypothesized to be due to a reduced conductivity of the alkali-soluble protein fraction when derived from pulp rather than whole biomass

Closed life-cycle aquaculture of sea lettuce (Ulva fenestrata): performance and biochemical profile differ in early developmental stages

Sea lettuce (Ulva) aquaculture has increased the last decade due to high productivity, wide environmental tolerance, and interesting functional and nutritional properties of the crop. Research focus has mainly been on adult biomass production, but knowledge of performance and biochemical content of early developmental stages – which are the basis to any large-scale production - is still limited. The life-history of Ulva alternates between a diploid sporophytic life-stage and a haplontic gametophytic life-stage. Whereas the sporophyte give raise to recombinant gametophytes through zoids, gametophytes can give raise to parthenogenetically developing, clonal gametes in absence of a mating partner. This study shows that recombinant gametophytes have a faster ontogenetic development, higher growth rate, as well as higher protein, fatty acid, and pigment contents compared to clonal gametophytes of the crop Ulva fenestrata. Nutrient addition is required for a normal development, but temperature and swarmer density have relatively small effects on the hatchery success, relative growth rate and biochemical profile of the juvenile biomass. Our study reveals that the selection of the life-history-phase in novel sea lettuce crop strains could largely contribute to the emerging seaweed aquaculture sector

New records from the southern North Sea and first records from the Baltic Sea of Kornmannia leptoderma

Combined genetic, morphological and ontogenetic observations show that the circumarctic boreal green algal macrophyte Kornmannia leptoderma has expanded its distribution range into the Baltic Sea, on a German coastal section of 220 km length. The species is also again (or still) established at its former extreme southern distribution limit in the North Sea, the German island of Helgoland, where it has not been detected during the last four decades. Macroscopic visible sporophytes of K. leptoderma are nowadays present in the Baltic Sea and at Helgoland from February to September, while they were in the past only detected from February to May at Helgoland. This capacity for formation of sporophytes in summer correlates with the circumstance that K. leptoderma from the Baltic Sea can complete its life cycle at 15°C while several studies conducted decades ago with material from Helgoland and from Pacific coasts consistently reported an inhibition of the algal gametogenesis at temperatures that exceed 12°C. Possibly K. leptoderma has undergone adaptations that facilitate its spread into warmer environments, unless the Kornmannia present in the Baltic Sea and on Helgoland today represents a newly introduced cryptic species