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DNA barcoding of the German green supralittoral zone indicates
the distribution and phenotypic plasticity of Blidingia species
and reveals Blidingia cornuta sp. nov.
Sophie Steinhagen,
1,2
Luisa Düsedau
1
& Florian Weinberger
1
1GEOMAR Helmholtz Centre for Ocean Research Kiel, Marine Ecology Department, Düsternbrooker Weg 20, 24105 Kiel, Germany
2Department of Marine Sciences-Tjärnö, University of Gothenburg, 452 96 Strömstad, Sweden
Address for correspondence: Sophie Steinhagen, sophie.steinhagen@gu.se
DOI https://doi.org/10.1002/tax.12445
Abstract 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 Helgo-
land (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 char-
acters. 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.
Keywords Baltic Sea; barcoding; Helgoland; Heligoland; phylogeography; ontogeny; tufA
Supporting Information may be found online in the Supporting Information section at the end of the article.
■INTRODUCTION
Green algae of the genus Blidingia Kylin are a substantial
component of the upper and mid-supralittoral zones of the
Northern Hemisphere, where they can be found as dense mats
on various natural and artificial surfaces and additionally as epi-
phytes on other macrophytobenthic organisms. Blidingia spp.
can withstand extreme conditions such as periodic and sus-
tained desiccation, heat waves or frost periods with snow cover,
and marine flooding as well as extended freshwater immersion.
Thus, representatives of the genus Blidingia often indicate the
transition between the marine or estuarine zone and the terres-
trial zone. Blidingia species shape these particular environments
and give them a unique and important ecological character by
providing habitats for small invertebrates. However, in-depth
understanding of the genetic species diversity within the
genus and hence about geographic species distribution is still
sparse. The recognition of Blidingia spp. is largely based on
morphological characters of mature thalli and ontogenetic
stages, while molecular knowledge is limited.
Kylin (1949) proposed the new genus Blidingia for En-
teromorpha minima Nägeli ex Kütz., based on observations
made by Bliding (1938). One of the main characteristics that
distinguishes Blidingia from the closely related and morpho-
logically similar genus Enteromorpha Link (nowadays Ulva L.)
is its small cells, with a diameter less than 10 μm (Kylin, 1949).
Both genera include species with monostromatic, tubular
thalli; however, their ontogenetic development was also found
to differ significantly (Kylin, 1949). The motile swarmers of
Blidingia have no eyespot, in contrast to the motile swarmers
of Ulva. The settled Blidingia swarmer grows into an elon-
gated tube that incorporates the intracellular spore contents,
and the empty spore sleeve is separated by a transverse mem-
brane. A prostrate disc develops, and from the expanded cen-
tre of this partly bi-layered disc, a monostromatic tube begins
to emerge (Bliding, 1938; Kylin, 1949). While several studies
Article history: Received: 29 Apr 2020 | returned for (first) revision: 12 Aug 2020 | (last) revision received: 5 Nov 2020 | accepted: 10 Nov 2020
Associate Editor: John Marinus Huisman | © 2021 The Authors.
TAXON published by John Wiley & Sons Ltd on behalf of International Association for Plant Taxonomy.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
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TAXON 00 (00) •1–17 Steinhagen & al. •DNA barcoding of German Blidingia species
SYSTEMATICS AND PHYLOGENY
supported these ontogenetic findings (Dangeard, 1961; Gayral,
1967), others emphasized variations in the ontogenetic deve-
lopment between different Blidingia species (Kornmann &
Sahling, 1978; Tatewaki & Iima, 1984; Iima, 1989), particularly
during spore sleeve formation (Bliding, 1963; Kornmann &
Sahling, 1978). Bliding (1963) stated, “the germinating tube
of the swarmer is mostly divided in a basal empty-cell and
an upper cell containing all the cytoplasm”, suggesting that
differences in spore sleeve formation have been observed.
Detailed observations of early ontogenetic developmental pat-
terns on spore sleeve formation were made by Kornmann
& Sahling (1978), who focused on the different Blidingia spe-
cies from Helgoland (Heligoland). Whereas half of the obser-
ved entities (B.minima (Nägeli ex Kütz.) Kylin, B.chadefaudii
(Feldmann) Bliding) cut off an empty spore sleeve (called
embryospore), this pattern was not observed for B.subsalsa
(Kjellm.) Kornmann & Sahling ex Scagel & al. and B.mar-
ginata (J.Agardh) P.J.L.Dang. ex Bliding. Thus, it was sug-
gested that early ontogenetic stages were suitable criteria for
species delimitation (Kornmann & Sahling, 1978).
Three different ontogenetic developmental patterns of
prostrate discs were observed in studies focused on the sexual
reproduction and early development of Blidinga minima from
Japan (Tatewaki & Iima, 1984; Iima, 1989). The authors distin-
guished compact discs with short cells (D-type), open discs
with longer cells (F-type) and an intermediate disc type (M-
type) (Iima, 1989). Notably, European specimens that exhib-
ited thallus discs similar to the F-type were assigned to a
newly differentiated species, namely B.chadefaudii (Bliding,
1938; Chadefaud, 1957; Kornmann & Sahling, 1978). How-
ever, due to the interfertility of specimens forming the different
disc-types identified by Iima (1989), it was suggested that
B.chadefaudii should be considered as a variant of B.minima
(Woolcott & al., 2000). Those authors undertook a molecular
reassessment of the taxonomic affiliation of individuals ex-
hibiting different disc-morphotypes based on nuclear rDNA
ITS sequences. By showing that the allegedly ontogenetic cri-
teria that were used for species delimitation encompass an
integrated continuum of variation, together with the fact that
molecular analysis and interbreeding-experiments did not de-
monstrate a species boundary between Japanese B.minima and
B.chadefaudii, the authors concluded that the two taxa are
conspecific. Thus, the observations of Woolcott & al. (2000)
blur the boundary between B.minima and B.chadefaudii and
underline that morphological characters within most Ulvales
are highly variable and that molecular sequencing is needed
to delimit species boundaries.
Currently, six Blidingia species are accepted taxonomi-
cally: B.chadefaudii,B.dawsonii (Hollenb. & I.A.Abbott)
S.C.Lindstr. & al., B.marginata,B.minima,B.subsalsa,B.tuber-
culosa (P.J.L.Dang.) Benhissoune & al. (Guiry & Guiry, 2020).
However, molecular knowledge of these species is sparse and
most have primarily been distinguished by morphological and
ontogenetic traits.
The presentstudy aimed to examine the diversity of Blidin-
gia species in the northern German State of Schleswig-Holstein.
Schleswig-Holstein’s coastline is of limited length, but is struc-
turally very diverse, including a south-east section of the fully
marine North Sea and a south-west section of the brackish Bal-
tic Sea, as well as various estuaries and lagoons. The area also
includes the North Sea island of Helgoland, the focus of phyco-
logical research in the mid-19th century (Reinke, 1889) and
among the best-studied seaweed habitats in Europe (Bartsch &
Kuhlenkamp, 2000). Long-term observations of the benthic flora
of Helgoland have shown that the establishment of artificial
substrata resulted in an increased abundance of Blidingia spp.
(Bartsch & Kuhlenkamp, 2000). Subsequent to C.W. Nägeli
collecting the holotype of B.minima on Helgoland around the
mid-19th century (historically: Enteromorpha minima,suppl.
Fig. S1), the first recordings of other Blidingia species for the
island were made by Wollny (1881). More recently, Kornmann
& Sahling (1978) documented the Blidingia species of Helgo-
land in a detailed synopsis, focusing on their developmental dif-
ferences. The study emphasized the presence of four Blidingia
species (B.chadefaudii,B.marginata,B.minima,B.subsalsa).
The authors observed several –and often vague –micro-morpho-
logical differences between the four species, but also one com-
monly shared macro-morphological character: All species were
characterized as unbranched tubes (Kornmann & Sahling, 1978).
According to species inventories, only B.minima and
B.marginata have been recorded from Schleswig-Holstein’s
mainland coasts (Schories & al., 2009). However, due to the
aforementioned taxonomic difficulties, historical records of
Blidingia species require confirmation, and the first molecular
assessment of the distribution of Ulvales and Ulotrichales in
the region (Steinhagen & al., 2019a,b) has indicated that the
present distribution of Blidingia spp. may not accord with
recent inventories. However, there has not been a molecular
characterization of the northern European Blidingia species
or a review of their allegedly significant identification criteria
undertaken until now.
A crucial point for molecular studies using DNA barcod-
ing is always the choice of suitable marker genes that are on
the one hand side variable enough to delimit species but also
stable within the respective species group. A marker gene that
has been widely used within molecular studies of green
algae is the coding gene for the ribulose-bisphosphate car-
boxylase large subunit rbcL (Saunders & Kucera, 2010). Later,
the plastid encoded tufA gene was promoted as the most suit-
able marker for delimitation of green algae, providing the high-
est amplification success and the largest barcode gaps for
green macroalgae (Saunders & Kucera, 2010). Genetic data-
bases such as GenBank include numerous sequences for both
markers. Reference sequences, however, need to be selected
with caution, as type specimens of Ulvales have rarely been
successfully sequenced.
By combining tufA and rbcL sequencing with morpholog-
ical and ontogenetic observations, we have assessed the diver-
sity of Blidingia species within Schleswig-Holstein and have
recognized an undescribed new species that is relatively abun-
dant in the area. In addition, our study highlights past difficul-
ties in distinguishing Blidingia species.
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Steinhagen & al. •DNA barcoding of German Blidingia species TAXON 00 (00) •1–17
■MATERIALS AND METHODS
Field collection and sample preparation. —Sites along
the North Sea and Baltic Sea coasts of Schleswig-Holstein –
including the island of Helgoland –were repeatedly visited
in the years 2014–2017 (see also Steinhagen & al., 2019a). In
2016, sampling also covered the heavily trafficked Kiel Canal,
which connects both sea areas (Fig. 1, sites 14–16, see also
Steinhagen & al., 2019b). Upper littoral and supralittoral zones
were checked for macroalgal growth with a focus on freshwater
inflows (e.g., drainages, river inflows, beach showers). Several
sites were re-visited in the years 2018 and 2019, to verify the
presence of populations and obtain material for cultivation
(Table 1). Altogether, Blidingia spp. were observed at 19 of
the visited sites (Fig. 1; Table 1).
Specimens were collected, placed into sealed plastic bags,
and stored on ice until further processing in the lab. Most sam-
ples were preserved as herbarium vouchers (see Table 1) and
lodged in the Natural History Museum Denmark, Copenha-
gen (C). A subsample was divided, with part of it stored at
4Cor−20C for subsequent morphological observation and
part of it stored in a microreaction tube at −80C for genomic
DNA extraction. Light microscopy observations were under-
taken, and microscopic documentation was carried out on
the remaining part, using a digital camera (Nikon DS-Vil)
attached to a light microscope (Nikon Eclipse TS 100). Salin-
ity was measured using a WTW portable conductivity meter
(Xylem Analytics, Weilheim, Germany).
In addition to the field collected samples, herbarium spec-
imens in the Herbarium of the Helgoland Biological Station of
the Alfred Wegener Institute (BRM) were included in our ana-
lyses (barcode numbers: BRM007967 and BRM008079; see
also Table 1). Several additional herbarium specimens were
investigated, including the type specimen of B.minima (Natu-
ralis Biodiversity Center, Leiden, Netherlands [L], barcode
L 0054691; suppl. Fig. S1). However, they yielded insufficient
amounts of DNA due to the vouchers being pre-treated with
fixation solutions, or the amplification of marker genes was
impossible due to impurities of the herbarium vouchers (dia-
toms, multi-algal vouchers, etc.). Thus, these herbarium vou-
chers were excluded from the molecular analyses of this study.
DNA extraction, amplification and sequencing. —
Genomic DNA was isolated from the lyophilized algal tissue
with an Invisorb Spin Plant Mini Kit (Stratec, Birkenfeld, Ger-
many) following the manufacturer’s protocol. Extracted DNA
was stored at −80C and used for amplification of the rbcL
and tufA genes. PCR amplifications of the rbcL gene used the
primer pairs rbcLstart and R750, as well as F650 and rbcLend
(Shimada & al., 2003). The PCR reactions were performed as
follows: 94Cfor1min;35cyclesat94
C for 30 s, at 56.3C
for 30 s, and at 72C for 1 min; and a final extension step at
72C for 7 min. PCR amplification of the tufA gene followed
the detailed description of Steinhagen & al. (2019a). PCR prod-
ucts were purified using the QUIAquick PCR Purification Kit
(Quiagen, Hilden, Germany). Subsequent sequencing of the
purified ampliconswas provided by GATC Biotech (Konstanz,
Germany). Forward and reverse sequence reads were assem-
bled to produce contigs in Sequencher (v.4.1.4, GeneCodes,
Ann Arbor, Michigan, U.S.A.), and a multiple sequence align-
ment was constructed for each gene region using MAFFT
v.7.402 (Katoh & al., 2002) (for Alignments, see supplemen-
tary Appendices S1 and S2). Sequences obtained in this study
are publicly available in GenBank (for accession numbers, see
Table 1).
Phylogenetic analyses. —RbcL and tufA sequences were
analysed in separate datasets. Newly generated sequences
Fig. 1. Sites of the Blidingia samples in northern Germany processed in this study. Overview map about northern Germany with numbered sampling
sites at the Wadden Sea (no. 1–10), on Helgoland (no. 11–13), within the Kiel Canal (no. 14–16) and in the Baltic Sea (no. 17–19).
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TAXON 00 (00) •1–17 Steinhagen & al. •DNA barcoding of German Blidingia species
Table 1. List of Blidingia samples collected and genetically assessed in 2014–2017 in northern Germany. Additionally, the herbarium (BRM) specimens included in this study are listed.
Species Sample ID Country Region Lat/Long
Station
Fig. 1 Date Collector Voucher
tufA
accession
no.
rbcL
accession
no.
Blidingia
marginata
S_129 Germany: Schleswig-
Holstein, Schluettsiel
Wadden Sea N 54.6813333
E 8.7544167
3 30 Jul 2014 S.Steinhagen C-A-99366 MH538542 –
Blidingia
marginata
S_147_A Germany: Schleswig-
Holstein, Pellworm
Wadden Sea N 54.49882
E 8.8087
4 31 Jul 2014 S.Steinhagen C-A-99378 MH475464 MN258044
Blidingia
marginata
S_147_B Germany: Schleswig-
Holstein, Schobuell
Wadden Sea N 54.5078167
E 8.9955667
6 31 Jul 2014 S.Steinhagen C-A-99379 MH538543 –
Blidingia
marginata
S_156 Germany: Schleswig-
Holstein, Woehrden
Wadden Sea N 54.1173167
E 8.9359333
9 05 Aug 2014 S.Steinhagen C-A-99382 MH538548 –
Blidingia
marginata
S_327 Germany: Schleswig-
Holstein, Heiligenhafen
Baltic Sea N 54.3765333
E 10.9800667
19 25 Aug 2014 S.Steinhagen –MH538549 –
Blidingia
marginata
S_474 Germany: Schleswig-
Holstein, Schluettsiel
Wadden Sea N 54.68435
E 8.75385
3 12 Sep 2014 S.Steinhagen C-A-99458 MH538546 –
Blidingia
marginata
S_577 Germany: Schleswig-
Holstein, Brunsbuettel
estuary
Wadden Sea N 53.889
E 9.101133
10 14 Apr 2015 S.Steinhagen –MH475465 MN258045
Blidingia
marginata
S_661 Germany: Schleswig-
Holstein, Nordstrand
Wadden Sea N 54.4707167
E 8.8068333
5 21 Apr 2015 S.Steinhagen –MH538544 –
Blidingia
marginata
S_708 Germany: Helgoland Helgoland N 54.1780333
E 7.8887167
11 23 Apr 2015 S.Steinhagen –MH538545 –
Blidingia
marginata
S_737 Germany: Helgoland Helgoland N 54.1825
E 7.8906167
12 24 Apr 2015 S.Steinhagen C-A-99417 MH538547 –
Blidingia
marginata
S_911 Germany: Schleswig-
Holstein, Aschau
Baltic Sea N 54.4608
E 9.92665
17 26 Aug 2017 S.Steinhagen –MN258036 MN258046
Blidingia
marginata
S_930 Germany: Schleswig-
Holstein, Schobuell
Wadden Sea N 54.50782
E 8.995567
7 27 Aug 2017 S.Steinhagen C-A-99681 MN258037 MN258047
Blidingia
marginata
S_941 Germany: Schleswig-
Holstein, Finkhaushallig
Wadden Sea N 54.41558
E 8.903633
8 29 Aug 2017 S.Steinhagen –MN258038 MN258048
Blidingia
marginata
S_944 Germany: Schleswig-
Holstein, Finkhaushallig
Wadden Sea N 54.41558
E 8.903633
8 29 Aug 2017 S.Steinhagen –MN258039 –
Blidingia
sp. 1
–Germany: Schleswig-
Holstein, Kiel Canal
Kiel Canal N 54.031933
E 9.300167
15 May 2016 S.Steinhagen –MG797655 –
Blidingia
sp. 1
–Germany: Schleswig-
Holstein, Kiel Canal
Kiel Canal N 54.369100
E 10.124917
16 May 2016 S.Steinhagen –MG797656 –
Blidingia
sp. 1
–Germany: Schleswig-
Holstein, Kiel Canal
Kiel Canal N 53.888778
E 9.116567
14 May 2016 S.Steinhagen –MG797657 –
Blidingia
sp. 1
S_21 Germany: Helgoland Helgoland N 54.1825
E 7.890617
12 23 Jul 2014 S.Steinhagen C-A-99667 MH538693 –
(Continues)
4Version of Record
Steinhagen & al. •DNA barcoding of German Blidingia species TAXON 00 (00) •1–17
Table 1. Continued.
Species Sample ID Country Region Lat/Long
Station
Fig. 1 Date Collector Voucher
tufA
accession
no.
rbcL
accession
no.
Blidingia
sp. 1
S_93 Germany: Schleswig-
Holstein, Aschau
Wadden Sea N 54.4608
E 9.92665
17 24 Jul 2014 S.Steinhagen –MH538691 –
Blidingia
sp. 1
S_179 Germany: Schleswig-
Holstein, Brunsbuettel
estuary
Wadden Sea N 53.889
E 9.101133
10 06 Aug 2014 S.Steinhagen C-A-99682 MH475459 MN258049
Blidingia
sp. 1
S_578 Germany: Schleswig-
Holstein, Brunsbuettel
estuary
Wadden Sea N 53.888778
E 9.116567
14 14 May 2015 S.Steinhagen –MN258043 –
Blidingia
sp. 1
S_622 Germany: Schleswig-
Holstein, Heiligenhafen
Baltic Sea N 54.3787167
E 10.95545
18 16 Apr 2015 S.Steinhagen –MH538692 –
Blidingia
sp. 1
S_813 Germany: Schleswig-
Holstein, Friedrich-
Wilhelm-Luebke-Koog
Wadden Sea N 54.83735
E 8.6122
1 24 Jul 2017 S.Steinhagen C-A-99677 MH475458 MN258050
Blidingia
sp. 1
S_815 Germany: Schleswig-
Holstein, Finkhaushallig
Wadden Sea N 54.41558
E 8.903633
8 24 Jul 2017 S.Steinhagen C-A-99678 MH475457 MN258051
Blidingia
sp. 1
S_818 Germany: Schleswig-
Holstein, Husum
Wadden Sea N 54.47113
E 9.027917
7 24 Jul 2017 S.Steinhagen C-A-99679 MH475456 –
Blidingia
sp. 1
S_828 Germany: Schleswig-
Holstein, Schobuell
Wadden Sea N 54.50782
E 8.995567
6 24 Jul 2017 S.Steinhagen C-A-99680 MH475455 MN258052
Blidingia
sp. 2
Hel_59 Germany: Helgoland Helgoland –ca. 11–13 25 Jul 1977 P.-H.Sahling BRM008079 MT076212 –
Blidingia
sp. 2
S_1 Germany: Helgoland Helgoland N 54.18367
E 7.888633
13 22 Jul 2014 S.Steinhagen C-A-99660 MH475461 –
Blidingia
sp. 2
S_34 Germany: Helgoland Helgoland N 54.18367
E 7.888633
13 23 Jul 2014 S.Steinhagen C-A-99668 MH475460 MN258053
Blidingia
sp. 2
S_39 Germany: Helgoland Helgoland N 54.1825
E 7.890617
12 23 Jul 2014 S.Steinhagen C-A-99671 MH475462 MN258054
Blidingia
sp. 2
S_124 Germany: Schleswig-
Holstein, Dagebuell
Wadden Sea N 54.73007
E 8.689167
2 30 Jul 2014 S.Steinhagen C-A-99663 MH475463 MN258055
Blidingia
sp. 2
S_949 Germany: Schleswig-
Holstein, Nordstrand
Wadden Sea N 54.4707167
E 8.8068333
5 29 Aug 2017 S.Steinhagen –MN258040 –
Blidingia
sp. 2
S_951 Germany: Schleswig-
Holstein, Nordstrand
Wadden Sea N 54.4707167
E 8.8068333
5 29 Aug 2017 S.Steinhagen –MN258041 MN258056
Blidingia
sp. 2
S_950 Germany: Schleswig-
Holstein, Nordstrand
Wadden Sea N 54.4707167
E 8.8068333
5 29 Aug 2017 S.Steinhagen –MN258042 –
Ulva
intestinalis
Hel_27 Germany: Helgoland Helgoland –ca. 11–13 29 Jul 1997 I.Bartsch BRM007967 MT076211 –
Specimens used in phylogenetic analysis are indicated by accession numbers in bold; however, all individuals were included in the calculation of intra- and interspecif ic genetic divergence values.
Version of Record 5
TAXON 00 (00) •1–17 Steinhagen & al. •DNA barcoding of German Blidingia species
were aligned with reference sequences downloaded from
GenBank and used for further phylogenetic analysis. Particu-
larly, sequences of specimens of the genera Ulva L., Korn-
mannia (Kjellm.) Bliding and Monostroma Thuret were inclu-
ded to assess relationships with closely related taxa, whereas
sequences of Protomonostroma undulatum (Wittr.) K.L.Vinogr.
(rbcL: HQ603387; tufA: HQ619275, MH475501) were cho-
sen as outgroups. Preference for reference sequence selection
was given to peer-reviewed sequences. The models that best
fit our data were found under the Akaike infor mation criterion
by employing MrModeltest v.2.2. (Nylander, 2004). For both
datasets, the optimal substitution model was determined and
found to be GTR+Γ+I. Maximum likelihood (ML) analyses
were then carried out using RAxML v.8 (Stamatakis, 2014),
employing the chosen substitution model with 1000 boot-
strap replicates for each alignment.
Cultivation. —Complete thalli of selected mature Blidin-
gia specimens were washed thoroughly and repeatedly with
sterile seawater (salinity of the respective collection site) to
remove dirt and adhering impurities and were isolated into
cultures. Clean thalli were transferred into polystyrene 24-
well plates and were incubated in sterile artificial seawater
adjusted to the salinity of the respective sites at 15C under
a photon flux density of 40–70 μmol m
−2
s
−1
and a 17 : 7 h
light : dark photo regime. To prevent the growth of diatoms,
1mgl
−1
GeO
2
was added. The thalli were examined daily
for sporulation events. After sporulation had taken place, adult
thalli were removed from the wells, and spore development
was observed with an inverted microscope (Nikon Eclipse
TS 100) and photographed (Nikon DS-Vil).
■RESULTS
Phylogeny. —The phylogenetic analyses performed on
datasets of the rbcL and tufA markers resulted in comparable
and nearly identical results, and almost equivalent evolution-
ary relationships of both investigated marker genes were dis-
covered (Fig. 2). The rbcL alignment consisted of a total of
695 positions (suppl. Appendix S1), whereas the tufA gene
dataset was 771 basepairs long (suppl. Appendix S2).
The node separating the genus Blidingia from closely
related and outgroup taxa received full bootstrap support for
both marker genes and unequivocably confirmed the taxo-
nomic position of Blidingia as a separate genus. Whereas the
tufA dataset reveals Ulva as sister clade to Blidingia (bootstrap
support: 99), the rbcL phylogram displays a reference sequence
of Kornmannia leptoderma (AF499677) as the closest relative
of Blidingia before Ulva. Both analyses resolve the German
Blidingia samples from the examined area in three clades
(Blidingia marginata,Blidingia sp. 1, Blidingia sp. 2) with high
(>85) to full bootstrap support (Fig. 2). The clades delimiting
B.marginata showed low intraspecific genetic variability
(tufA:0%–0.3%; rbcL:0%–0.2%) (see also Table 2) and could
be resolved with reference sequences (rbcL: HQ603379,
Canada; tufA: HQ610237, Canada). A reference sequence that
seemed to be incorrectly assigned to B.minima (MG721599)
clustered within the clade representing B.marginata.
The clusters representing Blidingia sp. 1 and Blidingia
sp. 2 could not be matched to any GenBank sequence entries.
However, both clades are clearly separated from other Blidingia
species and receive full bootstrap support (Fig. 2). Blidingia
sp. 1 finds its next relative in a well-delimited cluster of un-
identified Blidingia specimens from North America (HQ610241,
MF124265) (genetic dissimilarity of Blidingia sp. 1 and Bli-
dingia sp.: tufA 4.8%; rbcL n.a.), whereas the next closest rel-
ative of Blidingia sp. 2 is B.marginata (genetic dissimilarity
of B.marginata and Blidingia sp. 2: tufA 8.6%–9.4%; rbcL
3.2%–3.7%) (see also Table 2).
The phylograms of both the tufA (KT290281, HQ610239)
and rbcL genes (AF499676, MF90430, MF90429) include
clades of downloaded reference sequences from GenBank
that were supposed to represent Blidingia minima (Fig. 2).
However, the topology of these clades and thus their place-
ment within the trees is not congruent. One of the specimens
assigned to the clade representing B.minima within the tufA
phylogram originates from a site 30 km to the east in the
neighbouring German State of Mecklenburg-Vorpommern,
Wohlenberg, which was observed by us in a previous study
(Steinhagen & al., 2019a).
Notably, GenBank entries of the rbcL and tufA genes of
Blidingia minima showed significant differences in their
nucleotide reads (indicating different species are combined
in GenBank under this species name). Since most of the
uploaded sequences contain several ambiguous bases and
are rather short (250–500 bp), we decided to exclude them
from the displayed results. However, within this study, we
were not able to validate a genotype representing any of the
genotypes associated with B.minima in GenBank, not even
at the type locality of B.minima on Helgoland. Intra- and
interspecific divergence values of the here investigated Blidin-
gia entities are summarized and listed in Table 2.
Both herbarium specimens from Helgoland (Table 1)
showed no agreement between their previous identification
based on morphological traits and their molecular identifica-
tion by DNA barcoding. Within our phylogenetic analysis of
the tufA gene, voucher BRM008079 (morphologically identi-
fied as Blidingia minima, GenBank accession no.: MT076212)
clustered within the clade representing Blidingia sp. 2, and
voucher BRM007967 (morphological identity B.marginata,
GenBank accession no.: MT076211) was assigned to the clade
of Ulva intestinalis (Fig. 2).
Morphological characterization, habitat and distribu-
tion. —In the following section the Blidingia species present
in the study area of northern Germany are described in more
detail. Macro- and micromorphological observations includ-
ing respective ontogenetic features, as well as ecological and
distribution information, are presented for each species:
Blidingia marginata (J.Agardh) P.J.L.Dang. ex Bliding in
Opera Bot. 8(3) [Crit. Surv. Eur. Taxa Ulvales 1]: 32.
1963 ≡Enteromorpha marginata J.Agardh., Alg. Mar.
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Steinhagen & al. •DNA barcoding of German Blidingia species TAXON 00 (00) •1–17
Medit.: 16. 1842 ≡Enteromorpha nana var. marginata
(J.Agardh) V.J.Chapm. in J. Linn. Soc. London, Bot. 55:
416. 1956 –Lectotype: FRANCE. Nice, Alpes-Maritimes,
France, 1842 (LD Herb. Agardh No. 14161).
Habitat,seasonality and distribution.–Blidingia margi-
nata is the most common Blidingia species within the study
area and has the widest distribution. It is abundant on Baltic
Sea and Wadden Sea coasts and at Helgoland (Table 1). This
species can be found in remote as well as anthropogenically
strongly impacted habitats (see also Steinhagen & al., 2019a,b)
and inhabits fully marine and brackish water ecosystems.
However, B.marginata was seldomly observed in water bodies
Fig. 2. Comparative maximum likelihood phylograms of tufA and rbcL sequences from taxa of Blidingia from northern Germany. The grey-shaded
boxes indicate clades that were present in the study area and connects the respective clades of species in both phylogenetic analyses for direct com-
parison. Dashed boxes indicate the ambiguous use of the historic construct of “Blidingia minima”and highlight reference sequences identified as
such. Numbers at nodes indicate bootstrap values. Poorly supported nodes (<70% bootstrap support) are not labelled. Branch lengths are propor-
tional to sequence divergence. # symbol marks herbarium samples (see also Table 1).
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TAXON 00 (00) •1–17 Steinhagen & al. •DNA barcoding of German Blidingia species
below 5 PSU, and it was not detected in the Kiel Canal. As is
typical for the genus, B.marginata was observed growing as
dense turf mats in the upper and middle intertidal zone
(Fig. 3A–F).AdultsofB.marginata can be found through-
out the year, but their peak abundance was observed during
early and mid-summer (May–July). In late summer and fall
(August–October), dense stands began to bleach and popu-
lations shrank in size, so that in winter and spring only a
few smaller individuals were encountered. Individuals were
always found attached to the substratum and only observed
detached after extreme weather events. Especially in the North
Sea areas, B.marginata was the most abundant alga in the
intertidal zone. It occupied a variety of natural and artif icial
hard substrata (stones, cobble,breakwaters,woodenpiles,
etc.) and was often found growing epiphytically on other
macrophytes (e.g., Fuc us spp.) and higher plants (Phragmi-
tes sp.) in the intertidal (Fig. 3B,C). The same distribution
patterns were encountered in the Baltic Sea. Here, however,
B.marginata was found in mixed standswith Ulva intestina-
lis, and in some cases, young individuals of U.intestinalis
were difficult to distinguish from B.marginata by morpho-
logical characters alone. Individuals of B.marginata could
resist strong UV-radiation and desiccation in summer as
well as snowcover and long frost periods in winter (Fig. 3F).
Morphology.–Individuals of Blidingia marginata exhib-
ited all the morphological features described for this species
(Bliding, 1963), but also some differences. The long (1–12 cm)
and narrow thallus was tubular and usually formed dense mats
(Fig. 3D–F). Broader thalli were often wrinkled and twisted
and resembled Ulva intestinalis (Fig. 3G–I). Contrary to the
observations of Bliding (1963), who stated that specimens
of B.marginata exhibited rare small proliferations, 80% of
the individuals of the investigated material had microscopi-
cally visible branches or branchlet-like appendages (Fig. 3H).
These branchlets were often uni- or biseriate with a single api-
cal cell. Macroscopic branching in the middle or apical thallus
parts was rarely observed, whereas some specimens had mac-
roscopic branches in the rhizoidal zone. In young, narrow
thalli, the cells were arranged in distinct rows (Fig. 3J,K); how-
ever, mature or broader thalli exhibited only short cell rows or
no cell organization. Cells were quadratic to rectangular, some-
times of amorphous shape, 4–10 μm long and 2–9μm wide in
surface view. The chloroplast filled the whole cell with one
central pyrenoid (Fig. 3J,K).
Ontogeny.–As also described by Kornmann & Sahling
(1978), the quadriflagellate spore settled on the substratum
and immediately developed a germination tubewithout cutting
off an empty cell (Fig. 4A,B). The first mitotic cell divisions
resulted in the formation of a prostrate disc (Fig. 4C,D), which
then in most of the cases became 2-layered (Fig. 4E). The tubus
typically started to develop from lateral initial cells and was
rarely observed to develop form the disc’s centre.
Blidingia sp. 1
Habitat and distribution.–Specimens of this species
were also found on Baltic Sea and Wadden Sea coasts and
on Helgoland (Table 1). However, dense, turf-like populations
of Blidingia sp. 1 were not as frequent and abundant as those
of B.marginata, and they were more clearly restricted to the
upper supralittoral zone. Notably, most of the observed popula-
tions of Blidingia sp. 1 grew in the direct vicinity of freshwater
inflows (drain pipes, beach showers, stream run-off, etc.;
Fig. 5A). As a consequence –andincontrastwithB.margi-
nata and Blidingia sp. 2 –specimens of Blidingia sp. 1 were
rarely found desiccated during the summer months, despite
their location in the upper supralittoral. When freshwater in-
flows were more located towards the medio- or infralittoral,
Ulva intestinalis was the prevailing species, and Blidingia
sp. 1 was absent. The species was also absent from more
inland freshwater inflows that had no direct connection to
the sea. Blidingia sp. 1 was present throughout all seasons;
however, it was more common during July to August.
Table 2. Overview on intra- and interspecif ic divergence values of the Blidingia entities investigated within this study.
Interaction tufA [% difference] rbcL [% difference]
Intraspecific Blidingia marginata 0–0.3 0–0.2
Blidingia sp. 1 0–0.6 0
Blidingia sp. 2 0–0.2 0–0.2
Interspecific Blidingia marginata :Blidingia sp. 2 8.6–9.4 3.2–3.7
Blidingia marginata :Blidingia sp. 1 15.6–17.9 3.2–5.6
Blidingia sp. 1 : Blidingia sp. 2 11.8–13.1 6.4–6.8
Blidingia marginata :“Blidingia minima”13.7–15.4 4.1–4.4
Blidingia sp. 1 : “Blidingia minima”10.7–12.1 5.8–6.0
Blidingia sp. 2 : “Blidingia minima”10–11.1 4.7–5.7
Blidingia sp. 1 : Blidingia sp. (MF124265, HQ610241) 4.8 n.a.
Values for interspecific comparison of “Blidingia minima”were calculated on the respective historically mis-identified clades represented in Fig. 2
(dashed box tufA;rbcL lower dashed box). Therefore, they are framed by double quotation marks within this table.
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Fig. 3. Morphology of Blidingia marginata specimens from Germany. Population of B.marginata growing on a breakwater (A), epiphytic on Fucus
vesiculosus (B) and Phragmites (C), on cobbles (D) and on sand (E). As a species of the upper intertidal zone, it can manage frost periods (F). Indi-
viduals mostly exhibit no macromorphological branches (G&H) but often have microscopic branchlets (Hi&ii) and can be of cork-screw-like
morphology (I). Cells usually exhibit one central pyrenoid and are arranged in longitudinal rows (J&K). —Photos by S. Steinhagen.
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Morphology.–The morphology of Blidingia sp. 1 shows
similarities with the description of Bliding’s (1963) “Bli-
dingia minima var. ramifera”(not validly published, type not
indicated), which was later “raised to species rank”by Gar-
bary & Barkhouse (1987; as “Blidingia ramifera”). “Blidingia
ramife ra”is currently regarded as a synonym of B.mar-
ginata (Guiry & Guiry, 2019). However, there are distinct
differences:
The small, tubular and branched thalli were compact and
reached 1–10 mm in length (rarely taller) (Fig. 5B,E) whereas
thalli of “Blidingia ramifera”were distinctly larger (Bliding,
1963; Garbary & Barkhouse, 1987). The width of the thallus
increased as it proceeded from the rhizoidal zone to the tip
(0.08–0.8 mm wide). Branches were antler shaped, uni- to
multiseriate and present across the whole thallus (Fig. 5B,C).
Unbranched individuals were rarely encountered. In addition
to the clearly formed branches, spine-like microscopic append-
ages (10–60 μm) were frequently found across the whole thal-
lus (Fig. 5E,G). Branches and microscopic appendices were
blunt-ended. Thalli grew in tufts that formed dense stands,
and several individuals were connected by their rhizoidal
zones. Cells formed clear, longitudinal rows (Fig. 5F) that
sometimes blurred in broader thallus areas of the apical region
(Fig. 5G), whereas no distinct cell arrangement was observed
in mature individuals of “B.ramifera”(Bliding, 1963; Garbary
& Barkhouse, 1987). Individuals with unordered cell arrange-
ments were observed infrequently. The cells were quadratic,
rectangular, often polygonal with blunt to rounded corners
and 3–8μm long and 2–8μm broad. The chloroplast f illed
the cell or was rarely parietal, with 1 (rarely 2) central pyre-
noid(s) (Fig. 5F,H).
Ontogeny.–After the quadriflagellate spore had settled, a
germination-tube began to form (Fig. 4F). The cell content
migrated through the germination-tube and formed a germi-
nating cell, detaching the initial spore sleeve and in most cases
also parts of the germination tube (Fig. 4G,H). By mitotic cell
divisions, a monostromatic, relatively open disc developed,
and its cells were not as dense as in other entities (Fig. 4I,J).
After becoming distromatic, the disc bulged out, and an erect
tube formed.
Molecular analysis.–The sequence divergence of
the clade representing Blidingia sp. 1 from other species
within the genus, in combination with morphological dif-
ferences, indicates that Blidingia sp. 1 is genetically and
Fig. 4. Ontogenetic development of German Blidingia spp. After the spore of B.marginata had settled, a germination-tube began to form (A). No
cutting-off of an empty cell was observed (B). A prostrate disc began to form (C–E) with densly packed cells in its centre (E). The early development
of Blidingia sp. 1 started with the formation of a germination-tube after the spore had settled (F). The spores cell content migrated through the ger-
mination tube and formed a germinating cell while detaching the empty initial spore sleeve (G&H). Open discs were formed (I&J). The settled
spore of Blidingia sp. 2 formed a small germination tube (K) that then divided into an empty initial cell and a cell containing the cell content (L).
A monostromatic disc with dense cell arrangement formed (M) that in its centre became distromatic (N&O). —Photos by L. Düsedau.
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morphologically distinct from other previously described
species within the genus Blidingia. We here describe this
new species as Blidingia cornuta:
Blidingia cornuta S.Steinhagen & F.Weinberger, sp. nov. –
Holotype: GERMANY. Brunsbüttel harbour, Schleswig-
Holstein, N 53.889E 9.101133, 6 Aug 2014, S.Stein-
hagen S_179 (C barcode C-A-99682).
Figures 4F–J&5A–H.
Species description.–Thalli tubular, light to dark green,
compressed, bearing antler-like uni- and multiseriate branches
across the whole thallus (rarely unbranched), with rounded
Fig. 5. Morphology of the holotype of Blidingia cornuta sp. nov. (“Blidingia sp. 1”). (A) Type locality of B.cornuta at the Elbe estuary in Bruns-
büttel, Germany. The thalli are growing on a stone (i) directly underneath a water drainage pipe permanently releasing freshwater. The individuals
exhibited small, tubular and antler-like branched thalli (B&C), and also microscopic, blunt-ended, spine-like appendages were frequently observed
across the whole blade (D&E). The cells formed clear, longitudinal rows (F) that sometimes blurred in broader thallus areas of the apical region
(G). The cells were quadratic, rectangular, often polygonal with blunt to rounded corners, and the chloroplast filled the cell (rarely parietal) and con-
tained 1 (rarely 2) central pyrenoid(s) (F&H). —Photos by S. Steinhagen.
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intact branch apices, attached by rhizoids to substratum,
1–10 mm (mean ± 6 mm; rarely >1 cm) long, 0.08–0.8 mm
(mean ± 0.3 mm; rarely >1 mm) broad. Main axis increasing
in breadth from 10–35 μm (rhizoidal zone) to 0.3 mm (middle
and apical thallus). Middle thallus often with spine-like
appendices (20–35 μm in length), these appendices shorter
than real branches. Cells in surface view in clear longitudi-
nal rows in basal and middle thallus parts, short longitudi-
nal rows in apical regions, square, rectangular or polygonal
with blunt to round corners, most commonly 3–8μmlong
and 2–8μm broad. The chloroplast is cell-f illing (rarely pari-
etal covering most of the cell wall) and cells containing
1 (rarely 2) central pyrenoid(s). Reproduction by quadrifla-
gellate spores. From the spore a germination-tube arises, and
the cell contents migrate to a germinating cell, detaching the
initial spore sleeve and germination-tube. By mitotic cell divi-
sions a monostromatic disc develops, and after becoming dis-
tromatic, the disc bulges out and an erect tube is formed.
Etymology.–The species epithet cornuta (“horned”in
Latin) refers to the antler-like morphology of branches.
GenBank accessions.–MH475459 represents the sequence
of the tufA marker gene, and MN258049 is the respective
rbcL sequence.
Type locality.–Brunsbüttel harbour, Schleswig-Holstein,
Germany (N 53.889E 9.101133). Thalli were growing as
dense turf in the high intertidal zone directly under a drainage
with constant freshwater seepage from land on a bulkhead
(Fig. 5A). The site is part of the Elbe estuary, close to the out-
let of the Kiel Canal.
Other selected specimens examined (paratypes).–
Aschau, Schleswig-Holstein, Germany (N 54.4608;E
9.92665), 24 Jul 2014, S.Steinhagen S_93, GenBank MH
538691 (tufA), Baltic Sea; Heiligenhafen, Schleswig-Hol-
stein, Germany (N 54.3787167; E 10.95545), 16 Apr 2015,
S.Steinhagen S_622, GenBank MH538692 (tufA), Bal-
tic Sea; Helgoland, Germany (N 54.1825; E 7890617),
23 Jul 2014, S.Steinhagen S_21, GenBank MH538693
(tufA), North Sea; Schobuell, Schleswig-Holstein, Germany
(N 54.50782; E 8.995567), 24 Jul 2017, S.Steinhagen
S_828, GenBank MH475455 (tufA) and MN258052 (rbcL),
Wadden Sea; Husum, Schleswig-Holstein, Germany (N
54.47113; E 9.027917), 24 Jul 2017, S.Steinhagen S_818,
GenBank MH475456 (tufA), Wadden Sea; Finkhaushallig,
Schleswig-Holstein, Germany (N 54.41558; E 8.903633),
24 Jul 2017, S.Steinhagen S_815, GenBank MH475457 (tufA)
andMN258051(rbcL), Wadden Sea; Friedrich-Wilhelm-
Luebke-Koog, Schleswig-Holstein, Germany (N 54.83735;
E8.6122
), 24 Jul 2017, S.Steinhagen S_813,GenBank
MH475458 (tufA) and MN258050 (rbcL), Wadden Sea;
Brunsbuettel estuary, Schleswig-Holstein, Germany (N
53.893567; E 9.141733), 14 May 2015, S.Steinhagen
S_578, GenBank MN258043 (tufA), Wadden Sea/river Elbe.
Blidingia sp. 2
Habitat and distribution.–This entity was only observed on
Helgoland and in the northeastern Wadden Sea (Table 1) and
was not present in the Baltic Sea. It inhabited the upper supra-
littoral zone and was found growing as turfs, but more often it
was observed as small patches on stones, concrete, wooden piles
or other hard substrates (Fig. 6A,B). When growing epiphytic
on macrophytobenthic species (e.g., Fucus spp.), Blidingia
sp. 2 did not cover the host like B.marginata. Instead, single
individuals were found to be scattered across the host plants.
Morphology.–The morphology of Blidingia sp. 2 shows
strong similarities with the description of Kützing’s(1849)
Enteromorpha minima, which was later raised to the type of Bli-
dingia (as B.minima) by Kylin (1949). Striking morphological
congruities among Blidingia sp. 2 and B.chadefaudii from Hel-
goland (Kornmann & Sahling, 1978) are also obvious.
The thalli of Blidingia sp. 2 were mostly only few milli-
metres long (rarely taller than 1 cm) and 50–300 μmwide(sin-
gle individuals had broader thalli up to 700 μm) (Fig. 6C–E).
No branches in the middle or apical thallus parts were observed,
however the base sometimes exhibited branches (Fig. 6D,E).
Thalli were most often compressed, but inflated individuals
were alsopresent. Whereas cells form clear and distinct longitu-
dinal rows in the basal thallus parts (Fig. 6F), the arrangement
of cells is less organised in the middle and apical thallus parts
(Fig. 6G). Cells were of various shapes, quadratic to polygonal
with rounded corners, 4–8μmlongand4–6μmwideinsurface
view. No thickened cell walls, nor any lamellar internal struc-
tures were observed. The chloroplast was parietal or filled the
cell, with one central pyrenoid (Fig. 6H).
Ontogeny.–The main ontogenetic patterns discovered in
this study agree with the developmental descriptions of Blidin-
gia chadefaudii (rather than B.minima) made by Kornmann
& Sahling (1978). From the attached quadriflagellate spore,
a small germination tube was formed (Fig. 4K). The tube
divided into an empty initial cell and an upper cell containing
the cell contents (Fig. 4L). A monostromatic disc with a dense
cell arrangement was formed (Fig. 4M,N), which became dis-
tromatic in its centre (Fig. 4N,O) and gave rise to an erect
tube.
Molecular analysis.–Our molecular analysis demon-
strates that Blidingia sp. 2 is distinct from other Blidingia spe-
cies represented in GenBank. However, as described above,
morphological (Kützing, 1849; Kylin, 1949; Kornmann & Sah-
ling, 1978) as well as ontogenetic (Kornmann & Sahling, 1978)
patterns are in accordance with previously described entities.
■DISCUSSION
We here provide a strongly revised picture of the diversity,
distribution and morphological variability of Blidingia spp. in
Schleswig-Holstein, Germany, a study area that includes coas-
tal sections of two important ecosystems in northern Europe,
the North and Baltic Seas (Fig. 1). In total, 30 populations
of Blidingia spp. at 19 sites were analysed to cover the full
diversity in the study area. However, whereas older studies
and recent species inventories mention four Blidingia species
as abundantly present along the coastlines of Schleswig-
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Fig. 6. Morphology of Blidingia sp. 2. Typical habitat of Blidingia sp. 2 at the German peninsula Nordstrand (A&B). Individuals were found grow-
ing in patches and turfs on wooden piles (A) and stones of breakwaters (B). The thalli were unbranched in the middle and apical thallus region
(C&D); however, branching in the basal parts was observed infrequently (E). Cells of the basal part proceeded in clear and distinct longitudinal
rows (F), but the arrangement of cells is disturbed in the middle and apical thallus parts, and no clear str ucturewas observed (G). Cells were quadratic
to polygonal with rounded corners, and the chloroplast was found to be parietal or cell filling and one central pyrenoid was observed (H). —Photos
by S. Steinhagen.
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TAXON 00 (00) •1–17 Steinhagen & al. •DNA barcoding of German Blidingia species
Holstein (Kornmann & Sahling, 1978; Schories & al., 2009),
we only discovered three. Our findings suggest that this dis-
crepancy results from the exclusive use of morphological traits
and ontogenetic developmental characters as identification cri-
teria in the past. These criteria were probably invalid in part,
due to morphological variability within species. That morpho-
logical identification criteria for Blidingia species are not in
concomitance with more clear-cut molecular identification cri-
teria should be discussed in detail.
Of the four Blidingia species listed for Schleswig-
Holstein, only B.marginata was frequently observed. Blidin-
gia marginata was found to be the dominant species within
the intertidal zone, and it was frequently observed at Baltic
Sea and Wadden Sea coasts and on Helgoland. The species
is represented by a well-delimited clade with low intraspecif ic
variation (0%–0.3% tufA and 0%–0.2% rbcL) within both
provided phylogenetic trees (Fig. 2) and was well resolved to
the species level by including several peer-reviewed refe-
rence sequences (GenBank accessions HQ610237 tufA and
HQ603379 rbcL). Its closest relative was Blidingia sp. 2,
and both entities had a sequence dissimilarity of 8.6%–9.4%
within the tufA gene and 3.2%–3.7% in their rbcL sequences
(Table 2). However, the combination of morphological and
molecular techniques revealed that B.marginata exhibited a
larger morphological variability than expected (Fig. 3).
Individuals of Blidingia marginata exhibited all the mor-
phological features described for this species (Bliding, 1963),
and our ontogenetic observations (Fig. 4) are in accordance
with previous findings (Bliding, 1963; Kornmann & Sahling,
1978). The tubular thalli where either thin and elongated
or had a corkscrew-like morphology, and their cells were
arranged in clear longitudinal rows. It should be noted that
mature thalli are morphologically similar to small Ulva intes-
tinalis and could easily be confused with that species. Corre-
spondingly, a herbarium sample from Helgoland that was in
concordance with the morphological characters described
for B.marginata (Biological Station Helgoland Herbarium
BRM007967) was identified as U.intestinalis by DNA bar-
coding (Fig. 2).
Contrary to the observations of Bliding (1963), who
stated that specimens exhibited rare small proliferations, more
than 80% of the individuals of the examined material had
microscopic branches or branchlet-like appendages (Fig. 3H).
Bliding (1963) assigned specimens with branchlet-like struc-
tures to Blidingia marginata subsp. subsalsa (Kjellm.) Bliding.
Later, Scagel & al. (1989) raised B.marginata subsp. subsalsa
to species level, as already suggested by Kornmann & Sah-
ling (1978). Our observations indicate that B.marginata can
exhibit various morphologies, including those assigned to
B.subsalsa; however, such morphological differences were
not reflected by molecular differences of the marker genes tufA
or rbcL, and no delimitations of the different morphotypes
within our phylogenetic analyses were observed (Fig. 2).
Kornmann & Sahling (1978) observed that cultivated
swarmers, released by material identified as Blidingia subsalsa,
did not grow into the “naturally looking wildtype forms”of
B.subsalsa and that their first-generation offspring were
macro-morphologically rather identical with individuals of
B.marginata. Based on our results we conclude that the mor-
phological spectrum of B.marginata is broader than pre-
viously expected, as it includes the morphologies assigned
to B.subsalsa.
Based on literature (Kornmann & Sahling, 1978; Pankow,
1990; Schories & al., 2009), Blidingia minima was expected
to have the widest distribution in northern Germany. However,
our study revealed that this species is restricted to Helgoland
and some other North Sea locations. Past records, in particular
those from Baltic Sea locations, may represent misidentifica-
tions due to flawed historic species concepts.
This view is strongly supported by our analysis of avail-
able barcoding sequences. Screening of databases such as
GenBank for sequences of Blidingia minima provides several
hits for tufA and rbcL sequences of various length. However,
within our phylogenetic analyses (Fig. 2), different sequences
from around the globe and identif ied as B.minima fell in sev-
eral well delimited clusters (Fig. 2, dashed boxes) that are not
necessarily closely related or even cluster with B.marginata.
This unequivocally confirms that the recent species concept
of B.minima is ambiguous, combines genetically distinct enti-
ties, and needs clarification.
Even though sampling sites were chosen in a way that dis-
tances between the sites did not exceed 25 km (see also Stein-
hagen & al., 2019a,b), no genotypes in accordance with any
GenBank entries for Blidingia minima were observed in the
investigated area. One of the reference sequences allegedly
representing B.minima (KT290281) originates from Wohlen-
berg, 30 km to the east in the neighbouring German State of
Mecklenburg-Vorpommern and was observed in a previous
study. However, sequences from the type locality of B.minima,
Helgoland –several hundred kilometres away from Wohlen-
berg –, were so far missing, which gains importance when
we consider that this island was extensively studied within
our survey (Fig. 1). A frequently found entity on Helgoland
that could not be resolved to species level due to the absence
of any similar GenBank entries was Blidingia sp. 2 (Fig. 2).
Blidingia sp. 2 delimits in a unique clade and is the next clos-
est relative to B.marginata (Fig. 2, Table 2).
In addition to the above-mentioned molecular differences
of Blidingia sp. 2 and B.marginata, distinctive morphological
delimitations of the two entities were also observed. Blidingia
sp. 2 differed from specimens of B.marginata (Fig. 3) (Bli-
ding, 1963; Kornmann & Sahling, 1978) in generally exhibit-
ing smaller thalli and being mostly unbranched (Fig. 6). Only
few individuals exhibited macroscopic branching in the basal
thallus parts, and no microscopic branches were observed
(Fig. 6). Concurrently, the morphological features of adult Bli-
dingia sp. 2 (Fig. 6) showed high similarity with both the type
description of B.minima (Kützing, 1849; Kylin, 1949) and
traits described for B.chadefaudii (Kornmann & Sahling,
1978) from Helgoland.
Based on the results obtained within our study we
conclude: (1) The type locality of Blidingia minima (as
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Steinhagen & al. •DNA barcoding of German Blidingia species TAXON 00 (00) •1–17
Enteromorpha minima) is Helgoland (Kützing, 1849; Kylin,
1949); however, none of the entries in GenBank assigned to
B.minima could be verified on Helgoland. (2) The morpho-
logy (Fig. 6) of the well-delineated clade representing Bli-
dingia sp. 2 (Fig. 2) resembles the morphology described
for B.minima (Kützing, 1849; Kylin, 1949; Kornmann &
Sahling, 1978). (3) The molecular identification of a speci-
men within our study (Fig. 2, accession no.: MT076212) that
was identified as B.minima by P. Kornmann in 1977 (Table 1,
voucher number Herbarium Helgoland: BRM008079) is in
congruence with sequences of the entity representing Bli-
dingia sp. 2. (4) The ontogenetic development of Blidingia
sp. 2 (Fig. 4) is similar with that of B.chadefaudii des-
cribed in Kornmann & Sahling (1978). (5) We concur with
several authors who have highlighted the strong overlap
of morphological and ontogenetic traits of B.minima and
B.chadefaudii and thus suggested their conspecificity (Wool-
cott & al., 2000).
Based on these findings, the holotype of Blidingia mi-
nima cannot be critically identified for purposes of the precise
application of the name, and we suggest designating a speci-
men identified as Blidingia sp. 2 in our study as epitype of
B.minima.Blidingia minima, as we epitypify it here, has been
fully characterized molecularly (see Fig. 2), phenotypically
(see Results and Fig. 6), and ontogenetically (see Results
and Fig. 4) on the basis of isolates from the type locality of this
species, Helgoland (Kützing, 1849; Kylin, 1949).
■EPITYPIFICATION OF BLIDINGIA MINIMA
The holotype of Blidingia minima (L barcode L 0054691)
was collected at Helgoland, Schleswig-Holstein, Germany by
C.W. Nägeli. The exact date of the collection is not noted on
the herbarium voucher (suppl. Fig. S1). However, since the
work of Kützing (1849) refers to the herbarium voucher pre-
pared by Nägeli it can be expected that the holotype of
B.minima was sampled around the mid-19th century, which
is also in agreement with the biographical data of Nägeli
(1817–1891). The type specimen was first identified as
Enteromorpha minima and has been transferred to Blidingia
by Kylin in 1949 (Guiry & Guiry, 2019). Since no cultures
ex-type are available, it was re-collected at Helgoland and
cultivated (Figs. 4, 6). This collection is designated as epi-
type below. The described epitype is housed at C (C-A-
99668).
Blidingia minima (Nägeli ex Kütz.) Kylin in Förh. Kungl.
Fysiogr. Sällsk. Lund. 17: 181. 1949 ≡Enteromorpha mi-
nima Nägeli ex Kütz., Sp. Alg.: 482. 1849 ≡Enteromorpha
compressa var. minima (Nägli ex Hauck) Hamel in Rev.
Algol. 6(1): 65. 1931 ≡Enteromorpha nana var. minima
(Nägeli ex Hauck) Sjøstedt in Svensk Bot. Tidskr. 33: 38.
1939 –Holotype: GERMANY. Helgoland, Schleswig-
Holstein, exact collecting date unknown, Nägeli s.n.(Lbar-
code L 0054691!) –Epitype (designated here): GERMANY.
Helgoland, Schleswig-Holstein, N 54.18367E 7.888633,
23 Jul 2014, S.Steinhagen S_34 (C barcode C-A-99668).
Known geographical range.–This species was distrib-
uted on the German off-shore island Helgoland and at two
sites located at the German Wadden Sea (see Table 1).
Selected specimens examined.–Nordstrand, Schleswig-
Holstein, Germany (N 54.4707167; E 8.8068333), 29 Aug
2017, S.Steinhagen S_949, GenBank MN258040 (tufA),
Wadden Sea; Helgoland, Germany (N 54.17195; E 7.8993),
23 Jul 2014, S.Steinhagen S_34, GenBank MH475460
(tufA) and MN258053 (rbcL); Helgoland, Germany (N
54.18367; E 7.888633), 22 Jul 2014, S.Steinhagen S_1,
GenBank MH475461 (tufA); Helgoland, Germany (N 54.1825;
E 7.890617), 23 Jul 2014, S.Steinhagen S_39, GenBank
MH475462 (tufA) and MN258054 (rbcL); Dagebuell,
Schleswig-Holstein, Germany (N 54.1825; E 7.890617),
30 Jul 2014, S.Steinhagen S_124, GenBank MH475463
(tufA) and MN258055 (rbcL).
Although individuals from Helgoland could be identified
as Blidingia chadefaudii (Kornmann & Sahling, 1978), we
do not include B.chadefaudii as a heterotypic synonym of
B.minima. With the displayed results, we cannot rule out the
existence of B.chadefaudii, a species exhibiting unique traits
like a thickened inner cell wall that presents a lamellar struc-
ture of parallel arranged riffles (Chadefaud, 1957). Also,
based on the multiple sequence entries in GenBank, it can
be assumed that the genus Blidingia harbours even more spe-
cies than we are aware of today. However, there is currently no
evidence of the presence in Germany of B.chadefaudii in the
past or today. The only existing records were based on material
that did not show the characteristic traits of B.chadefaudii,
such as cell wall thickenings (Kornmann & Sahling, 1978),
and were apparently due to a misinterpretation of life cycle
traits that are variable within B.minima.
Furthermore, Blidingia cornuta sp. nov. can be clearly
distinguished from other described species of Blidingia by a
unique combination of characters: Our phylogenetic analyses
of the tufA and rbcL marker genes clearly distinguished B.cor-
nuta from its closest genetic relatives. Within the tufA tree, a
clade that contained sequences of an undescribed entity orig-
inating from Manitoba, Canada (HQ610241) and from Alaska,
U.S.A. (MF124265) was found to be the next known closest
relative to B.cornuta. Due to significant sequence dissimi-
larities of 4.8%, the independent status of B.cornuta can be
confirmed (Fig. 2, Table 2). The clade representing B.cornuta
is also supported with maximum bootstrap values (Fig. 2).
Additionally, ontogenetic and morphological features sepa-
rate the newly proposed species from already existing spe-
cies (Fig. 4).
A unique pattern of ontogenetic development was ob-
served in individuals of Blidingia cornuta: After a clear ger-
mination-tube had begun to form (Fig. 4F), the cell con-
tents migrated through the germination-tube and formed a
germinating cell, detaching the initial spore sleeve and
germination-tube (Fig. 4G,H). This kind of pattern has not
been observed in any other described Blidingia species
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TAXON 00 (00) •1–17 Steinhagen & al. •DNA barcoding of German Blidingia species
(Kylin, 1949; Bliding, 1963; Kornmann & Sahling, 1978). As
another unique trait, thalli exhibited a specific antler-like
branching pattern, together with a cell arrangement in longitu-
dinal rows that has not previously been observed in any other
Blidingia species. It should be mentioned, however, that “Bli-
dingia minima var. ramifera”(Bliding, 1963) exhibits some
similarities –but also significant differences –with the newly
described B.cornuta. The thalli described by Bliding (1963)
were distinctly taller in size and reached a length of up to
50 cm, whereas thalli of B.cornuta are less than 1 cm in length
(rarely taller). The branches of “B.minima var. ramifera”were
described as elongate, while the branching pattern in B.cor-
nuta is antler-like, and the respective branches are compact
rather than elongate. Bliding (1963) reported that cells of
“B.minima var. ramifera”were predominantly unarranged or
only arranged in short rows, whereas most specimens of B.cor-
nuta have clearly visible longitudinal cell rows that are evident
throughout the thallus. Garbary & Barkhouse (1987) treated
“B.minima subsp. ramifera”at species level. Their description
of “B.ramifera”encompassed the same striking differences
from B.cornuta as previously described for “B.minima subsp.
ramifera”. Hence, branched specimens of “B.ramifera”were
regarded as morphotypes of B.marginata (Burrows, 1991;
Guiry & Guiry, 2019). However, the genetic distinction of
B.cornuta seems indeed reflected in a unique branching pat-
tern. In this light, the status of “B.ramifera”should be re-
evaluated based upon support with genetic markers. Several
unidentified Blidingia sequences are available via GenBank
that hint a hidden diversity that is still to be discovered. Our
study indicates once again that it can be rewarding to reassess
the exact taxonomic relationships of allegedly well-known
species groups within the Ulvophyceae based on molecular
markers and subsequent phylogenetic techniques to reveal
exact species relationships, potential morphological plasticity,
and species-specific ecological traits.
We can conclude that the historical species concepts for
several Blidingia spp. are flawed and problematic for species
occurring in northern Germany. Misinterpretation of pheno-
typic plasticity in mature thalli, and to some degree also in onto-
genetic developmental stages, has led to misidentifications
in the past, and species delimitation based on morphologi-
cal traits is often impossible. Thus, our findings support
the use of molecular methods for correct and clear species
identification and devalue the use of morphological charac-
ters alone.
■AUTHOR CONTRIBUTIONS
SSt: original concept, experimental design, fieldwork and algae
collection, laboratory work, macro- and microscopic observation, phy-
logenetic analysis, drafting and editing manuscript; LD: algae collec-
tion, laboratory work and algae cultivation; FW: algae collection,
original concept, drafting and editing manuscript. —SSt, https://orcid.
org/0000-0001-8410-9932; LD, https://orcid.org/0000-0002-2750-6437;
FW, https://orcid.org/0000-0003-3366-6880
■ACKNOWLEDGEMENTS
We would like to thank the herbarium of the Biological Station
Helgoland of the Alfred Wegener Institute, especially Dr. Inka Bartsch,
for providing us with material of the vouchers used in this study. We are
also thankful to the Naturalis Biodiversity Center, Leiden, the Netherlands
for their close cooperation and granting us access to their valuable voucher
collection of macroalgae. Additionally, we would like to express our
thanks to the Natural History Museum Denmark, Copenhagen for their
close cooperation and lodging the vouchers of this study. Furthermore,
we thank Joel White for the valuable comments he made on the
manuscript.
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