Molecular identification of the ubiquitous green algae Ulva reveals high biodiversity, crypticity, and invasive species in the Atlantic-Baltic Sea region

Abstract

Correct species identification is fundamental for assessment and understanding of biodiversity. Erroneous species
identification may impede conservation management and may delay detection of invasive species. The ubiqui-
tous green algal genus Ulva is known for its wide environmental tolerance, plastic morphology, occurrence of
cryptic species and ambiguous species concepts that hinder clear identification. We used molecular monitoring to
assess species diversity and distribution of Ulva along the full Atlantic-Baltic Sea salinity gradient (> 10,000 km).
Ulva specimens were collected from Denmark, Finland, Germany, Norway, and Sweden. DNA barcoding analysis
of the tufA gene revealed 20 genetic entities in total, of which 11 could be identified to species level
(U. californica, U. flexuosa, U. torta, U. linza, U. prolifera, U. fenestrata, U. australis, U. intestinalis, U. compressa,
U. gigantea, U. lacinulata). Nine entities (Ulva sp. 1–9; [Ulva capillata]) yielded novel sequence reads that
belonged to either unidentified species, species complexes, or singletons. At least 3 of the discovered species
(U. australis, U. californica, U. gigantea) are considered non-native and potentially invasive. Furthermore,
considerable differences between the observed and the historically estimated species distributions were found.
The highest diversity was recorded in the Atlantic and Skagerrak region whereas only two entities of taxo-
nomically accepted species where found north-east the Blekinge coast. Our study shows that the species diversity
of Ulva in the study area is diverging from previous reports, and that molecular methods are imperative for
species identification in this morphologically plastic genus. Furthermore, the presence of non-native species
indicates a necessity for further fine-scale monitoring in specific areas to e.g. mitigate formation of green tides.

Full text

Algal Research 73 (2023) 103132
Available online 5 May 2023
2211-9264/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Molecular identication of the ubiquitous green algae Ulva reveals high
biodiversity, crypticity, and invasive species in the Atlantic-Baltic
Sea region
Sophie Steinhagen
*
, Samanta Hoffmann, Henrik Pavia, Gunilla B. Toth
Department of Marine Sciences-Tj¨
arn¨
o, University of Gothenburg, SE-452 96 Str¨
omstad, Sweden
ARTICLE INFO
Keywords:
Ulva
Phylogeography
tufA
DNA barcoding
Invasiveness
Algal resources
ABSTRACT
Correct species identication is fundamental for assessment and understanding of biodiversity. Erroneous species
identication may impede conservation management and may delay detection of invasive species. The ubiqui-
tous green algal genus Ulva is known for its wide environmental tolerance, plastic morphology, occurrence of
cryptic species and ambiguous species concepts that hinder clear identication. We used molecular monitoring to
assess species diversity and distribution of Ulva along the full Atlantic-Baltic Sea salinity gradient (>10,000 km).
Ulva specimens were collected from Denmark, Finland, Germany, Norway, and Sweden. DNA barcoding analysis
of the tufA gene revealed 20 genetic entities in total, of which 11 could be identied to species level
(U. californica, U. exuosa, U. torta, U. linza, U. prolifera, U. fenestrata, U. australis, U. intestinalis, U. compressa,
U. gigantea, U. lacinulata). Nine entities (Ulva sp. 1–9; [Ulva capillata]) yielded novel sequence reads that
belonged to either unidentied species, species complexes, or singletons. At least 3 of the discovered species
(U. australis, U. californica, U. gigantea) are considered non-native and potentially invasive. Furthermore,
considerable differences between the observed and the historically estimated species distributions were found.
The highest diversity was recorded in the Atlantic and Skagerrak region whereas only two entities of taxo-
nomically accepted species where found north-east the Blekinge coast. Our study shows that the species diversity
of Ulva in the study area is diverging from previous reports, and that molecular methods are imperative for
species identication in this morphologically plastic genus. Furthermore, the presence of non-native species
indicates a necessity for further ne-scale monitoring in specic areas to e.g. mitigate formation of green tides.
1. Introduction
Invasions by plants, animals, and pathogens into non-native envi-
ronments is one of the most signicant threats to biodiversity [1].
Invasive species can increase the risk of extinction and inuence the
genetic composition of native populations, as well as change ecosystem
functioning by altering nutrient cycles, hydrology, habitat structure, and
disturbance regimes [2]. One important pathway for introduction of
invasive species is human trade with plant and animal species for
cultivation [3,4]. In aquatic environments, aquaculture has historically
caused both deliberate and accidental introductions of invasive species
including molluscs [5], crustaceans [6], sh [7,8], and seaweeds [9].
Today aquaculture is one of the fastest expanding sectors for cultivation
of new food species [10]. Recently, green macroalgae of the genus Ulva
attracted interest from the growing aquaculture industry [11–16] due to
their fast growth and high nutrition value [13,17–20]. However, these
traits also make them prone to introduction to new ecosystems by
human dispersal [21–23], and combined with the fact that some
opportunistic species can form green tides under suitable nutrient con-
ditions [24–27], correct species identication and phylogeographic as-
sessments of this taxonomic group is crucial before development as
aquaculture species.
After Linnaeus [28] formalised the binomial nomenclature and
described many of today's macroalgal species, Swedish phycological
pioneers like Agardh [29–32], Areshoug [33], Ahlner [34], and Kjell-
mann [35] laid an important basis for seaweed taxonomy by studying
the algal diversity of Scandinavia and adjacent regions of the Baltic Sea.
Present day taxonomic identication of green macroalgae in the
Atlantic-Baltic Sea region is mostly based on morphological characters
and several identication keys and inventory lists have been compiled
* Corresponding author.
E-mail address: sophie.steinhagen@gu.se (S. Steinhagen).
Contents lists available at ScienceDirect
Algal Research
journal homepage: www.elsevier.com/locate/algal
https://doi.org/10.1016/j.algal.2023.103132
Received 21 February 2023; Received in revised form 20 April 2023; Accepted 2 May 2023

Algal Research 73 (2023) 103132
2
(e.g. [36–45]). New species are being constantly described
[13,14,25,46–49] and allegedly well-dened species are revised
[27,49–55]. A striking example is the genus Ulva (e.g. [56]), which
currently comprises 85 taxonomically accepted species, >550 historic
species names, and several entities with unclear taxonomic status [57].
Ulva species exhibit a variety of complex morphologies
[25,27,36,53,58–61], and therefore, morphologically based species
identication often lead to mis-identication [25,27,50,52,53,56,61].
The increasing use of molecular techniques, such as DNA barcoding,
has led to signicant taxonomic revisions, especially within the orders
Ulvales and Ulotrichales [14,27,49,50,52–54,56]. However, previous
surveys using molecular methods for species identication in the
Atlantic-Baltic Sea region are relatively scarce and cover only small
areas (e.g. [27,53,55,61–63]). Therefore, the reported number of species
could be over- or under-estimated and non-native and potential invasive
species may remain undetected. These assumptions are strengthened by
studies of bordering areas, which show that species identication using
molecular methods differs from classical species lists based on
morphological characters [27,53].
The aim of this study was to present a rst molecular monitoring
(DNA barcoding of the tufA gene) to assess species diversity and distri-
bution of the ubiquitous and morphologically variable green algal genus
Ulva along the full Atlantic-Baltic Sea region. The Atlantic-Baltic Sea
region is characterized by a strong salinity gradient which stretches from
fully marine conditions in the Skagerrak (28–32 PSU), to almost fresh-
water in the Bothnian Bay (2–0 PSU) [64]. This impressive salinity
gradient directly affects the species distribution and diversity depending
on the salinity tolerance of different species [65,66]. As members of the
Ulva genus are mainly marine, we expect that the diversity to be overall
higher in the fully marine conditions and to decrease with the decreasing
salinity regimes. Similarly, we expect comparisons of recent molecular
data and historical inventory lists, which were based on morphological
identication criteria only, to be divergent due to the appearance of
cryptic species. Additionally, the substrate availability strongly differs
along the coastline of the Baltic Sea. While widespread rocky shores
provide seaweeds with hard substrate for attachment at the Swedish
west coast, a scarcity of hard substrata prevails along the coasts of the
Baltic proper where mainly sand and gravel beaches occur. These con-
ditions are also hypothesized to affect the species composition of Ulva,
since some species grow unattached, while others prefer to attach to a
hard substrate.
2. Material and methods
2.1. Study area, eld collection and sample preparation
Samples of the genus Ulva (n =1000) used in the present study were
collected along the full salinity gradient present in the Baltic Sea and
adjacent areas such as the Kattegat, Skagerrak, and the eastern North
Sea (Fig. 1). In total, 287 sampling sites, of which 121 in Sweden, 66 in
Denmark, 54 in Germany, 26 in Norway and 20 in Finland, were visited
during 2014–2021 (including different seasons, see also supplementary
Table S1). The measured salinity at the respective sites where Ulva
vegetation was occurring ranged from 3.5 to 36 PSU and is presented in
Fig. 1 (see also supplementary Table S1). In addition, both water tem-
perature (◦C) and oxygen levels (mg L
−1
) were measured at most of the
sites (Supplementary Table S1). Sampling was designed so that a variety
of habitats, such as rock pools, harbours, marine national parks, estu-
aries, fjords, drain channels as well as exposed and sheltered coastal
areas were included to reect the different ecosystems present in the
Baltic Sea area. Additionally, different substrates (organic and inor-
ganic, natural and articial) of the attached thalli were included and
drift populations were sampled as well. Algae collections to a depth of
~1.5 m below mean sea level in the supra- and midlittoral zones were
conducted using waders. In addition, samplings of the mid- and infra-
littoral zones of chosen sites were conducted via snorkelling.
At each site, representative specimens of each morphotype and all
observed populations were collected, ranging from the supralittoral to
the sublittoral. The sampling also included drifting and epiphytic spec-
imens. For DNA barcoding, clean and epiphyte-free tissue samples (~1
cm
2
) of representative individuals were collected and additional sam-
ples for morphological analyses were taken. During sampling, all sam-
ples were stored in a portable freezer (−20 ◦C) until transfer to −80 ◦C in
the laboratory.
2.2. Molecular and phylogenetic analysis
From lyophilized tissue of 1000 specimens of Ulva, genomic DNA
was extracted using the Invisorb Spin Plant Mini Kit (Stratec, Birkenfeld,
Germany) following the manufacture's protocol. Extracted DNA was
stored at −80 ◦C and used for the amplication of a portion of the tufA
(~ 770 bp) to identify specimens. PCR amplicons of the tufA gene were
generated for all specimens, following the detailed description available
in Steinhagen et al. [53]. The PCR products were rst assessed by
agarose gel electrophoresis and subsequently puried using the QUIA-
quick PCR Purication Kit, Quiagen (Hilden, Germany). Sanger
sequencing of the puried amplicons was conducted by Eurons Ge-
nomics (Konstanz, Germany). To produce contigs and rule out potential
sequencing errors forward and reverse sequence reads were assembled
in Sequencher (v. 4.1.4, Gene Codes Corporation, Ann Arbor, MI) and a
multiple sequence alignment was constructed using MAFFT [67]. All
sequences obtained in our study are publicly available in the genetic
database GenBank (for accession numbers consult supplementary table
S1).
By using the BLAST function in GenBank, rst identications based
on the specimens' tufA sequences were made. For resolving species
identities peer-reviewed and annotated reference sequences down-
loaded from GenBank were included in subsequently performed phylo-
genetic analyses. The identication of Ulva species followed the latest
taxonomic revisions by Hughey et al. [51,52]. Based on the multiple
sequence alignment an optimal substitution model (GTR+G+I) was
determined using MrModeltest software version 2.2 [68]. A maximum-
likelihood analysis was performed using RAXML (version 8; [69]) with
1000 bootstrap iterations. No additional species delimitation methods
were applied since a revision of the Ulva taxonomy and systematics was
beyond the scope of this study.
2.3. Morphological analysis
The focus of the present study was the molecular diversity and
phylogeographic distribution of Ulva spp. in the Atlantic-Baltic Sea
transect. However, morphological characters were also investigated and
recorded and some of the most striking ndings for selected Ulva species
and ecotypes are reported here in order to assist with future
identications.
Pre-identication of Ulva species was based on typical morpho-
anatomical characters (e.g. overall thallus morphology, cell form, cell
arrangement, number of pyrenoids per cell, etc.) and observations were
based on original diagnoses and identication criteria of identication
keys and previous studies [27,36,39,42,53,59,60,63,70–72]. Macro-
morphological characters were observed on fresh and frozen material
with a binocular microscope (Leica, Wetzlar, Germany) and micromor-
phological characters were observed with a microscope (ZEISS, Ober-
kochen, Germany) tted with a camera (Canon, Tokyo, Japan).
2.4. Comparisons of the recent and historic species inventory and
phylogeographic distributions
To compare the ndings of the present study with historic species
inventories and assess species´specic phylogeographic distribution
patterns, historical publications, inventories, and species keys of Ulva
spp. from the study area and neighbouring regions ([37–42,44,45]; see
S. Steinhagen et al.

Algal Research 73 (2023) 103132
3
Fig. 1. Map of sampling sites in the Atlantic Baltic-Sea transect and respective species distributions of Ulva spp.. (A) Overview map of the Atlantic-Baltic Sea transect
with respective sea surface salinity. Visualization of the salinity gradient within the Baltic Sea by isohalines with particular salinity values (PSU) in circles (HELCOM-
data) dropping with increasing distance from the North Sea. Red dots mark sample sites which represent the whole salinity gradient of the area. Insets B – I provide
the distribution of Ulva spp. in the Atlantic-Baltic Sea transect, genetically veried within this study. The distribution of (B) U. intestinalis and U. linza, (C)
U. compressa and U. prolifera, (D) U. lacinulata and U. torta, (E) U. exuosa and U. gigantea, (F) U. fenestrata [dark and light red], U. australis [dark red], and
U. californica [dark red], (G) Ulva sp. 2 [U. capillata] and Ulva sp. 3, (H) Ulva sp. 6 and Ulva sp. 8, and (I) Ulva sp. 1 (red dots), Ulva sp., 4 (orange dots), Ulva sp. 5
(yellow dots), Ulva sp. 7 (light green), Ulva sp. 9 (dark green), is presented. Full data is available in supplementary Table S1. (For interpretation of the references to
colour in this gure legend, the reader is referred to the web version of this article.)
S. Steinhagen et al.

Algal Research 73 (2023) 103132
4
also Table 2) were compared with molecular and morphological data
obtained in this study. To allow for detailed phylogeographic distribu-
tion patterns, the analysis in the present study also included data
collected during recent molecular assessments of the Ulva biodiversity
along the German coasts from Steinhagen et al. [27,53,61].
3. Results
3.1. Molecular species identication
A total of 1000 Atlantic and Baltic Ulva individuals were processed
genetically for species discrimination and identication, based on tufA
sequence data (Supplementary Table S1). The full dataset was subject to
phylogenetic analyses (see Supplementary table S1). To allow for a
condensed phylogram, an analysis with selected representatives was
also performed (Fig. 2, Table 1). All unique observed unidentied spe-
cies, species complexes, and singletons remained in the condensed tree
(Fig. 2, Table 1).
The phylogenetic analysis separated the investigated specimens into
20 taxonomic entities, where 11 entities could be resolved based on
peer-reviewed reference sequences (Ulva californica, Ulva exuosa, Ulva
torta, Ulva linza, Ulva prolifera, Ulva fenestrata, Ulva australis, Ulva
intestinalis, Ulva compressa, Ulva gigantea, Ulva lacinulata) provided by
GenBank. Nine clades did not match any of GenBanks reference se-
quences and several singletons were observed (Fig. 2, Supplementary
Table S1). It should however be mentioned that during the revision
process of this work the clade encompassing Ulva sp. 2 (Fig. 2, Supple-
mentary Table S1) was described as the new species Ulva capillata
Steinhagen [49].
All available tufA sequences of type material have been included in
Fig. 2. Maximum Likelihood phylogenetic tree of Ulva spp. tufA sequences present in the Atlantic-Baltic Sea gradient. The phylogram was rooted on Umbraulva
sequences. Coloured clades represent identied species found in the present study, whereas grey shaded clades represent unidentied Ulva species, species com-
plexes, and singletons. The clade indicated as Ulva sp. 2 has recently been described as Ulva capillata (38). Numbers at branches indicate bootstrap values >70.
Branch lengths are proportional to sequence divergence.
S. Steinhagen et al.

Algal Research 73 (2023) 103132
5
the phylogram (only reduced phylogram shown), it should however be
mentioned that only 4 clades - all foliose species - (U. fenestrata,
U. australis, U. gigantea, U. lacinulata) could be clearly identied based
on the presence of sequenced type material. Although, peer-reviewed
reference sequences have been used for the identication of other
clades to species level, the absence of sequenced type material of these
species might cause later taxonomic changes or re-naming when type
material sequences become available, and the application of names
should be carefully checked.
The phylogenetic analysis split the investigated taxa and singletons
into three main branches and the overall topology is in accordance with
previous studies (e.g., [25,51–53,73]). Most of the species clades ob-
tained full bootstrap support (Fig. 2).
The sequences identied as U. linza by reference sequences, clustered
in two separated clades. Even though the two clusters were only sepa-
rated by a genetic distance ranging from 1.3 to 2.1 %, the node sepa-
rating both clades received a bootstrap support value of 91 (Fig. 2).
Therefore, we will refer to these clades as the Ulva linza-complex.
3.2. Ulva spp. genetic diversity in the Atlantic-Baltic Sea region and
relative frequencies
The different Ulva spp. showed distinct distribution patterns within
the Atlantic-Baltic Sea region (Fig. 1). For most species and certain
ecotypes clear distribution patterns associated with prevailing salinity
regimes were observed. Highest diversity was observed in the Atlantic
and Skagerrak region (11 taxonomically valid species, 7 unidentied
species, species complexes, singletons), followed by the Kattegat (9
taxonomically valid species, 5 unidentied species, species complexes,
singletons) (Fig. 1). After passing the Danish straits, certain Ulva species
such as e.g. U. fenestrata were not recorded anymore in the Kiel bay (8
taxonomically valid species, 5 unidentied species, species complexes,
singletons) (Fig. 1). Foliose individuals recorded after passing the
Danish straits were U. gigantea, U. rigida, U. compressa, or U. intestinalis.
Table 1
List of Ulva samples collected in the Atlantic-Baltic Sea gradient and used in the displayed phylogenetic
tree (see Fig. 2; for full dataset see supplementary Table S1).
GenBank accession no. Species Voucher no. Collection date Country Collection Site Lat Lon
OL421370 Ulva australis NO_103 20200708 Norway Brekkestø 58.19468 8.346613
OL421395 Ulva australis NO_134 20200709 Norway Borshavn 58.10042 6.582716
OP267652 Ulva californica DK_114_A 20200721 Denmark Fredrikshavn_harbour 57.42541 10.52816
OL421402 Ulva compressa NO_144 20200709 Norway Egersund 58.43743 5.90904
OL421445 Ulva compressa SV_803 20200627 Sweden Tj¨
arn¨
o 58.88325 11.11783
OL421284 Ulva fenestrata DK_216 20200725 Denmark Middelfart_1 55.54396 9.768291
OL421396 Ulva fenestrata NO_135 20200709 Norway Borshavn 58.10042 6.582716
OL421428 Ulva fenestrata SV_786 20200625 Sweden Fj¨
allbacka_strand 58.59184 11.27837
OP267812 Ulva gigantea SV_159.1 20190402 Sweden Hamburgsund 58.5527 11.2683
OL421110 Ulva intestinalis DK_025 20200715 Denmark Vejby 56.10651 12.17557
OL421308 Ulva intestinalis F_31B 20200915 Germany Riems 54.18127 13.34739
OL421524 Ulva intestinalis SV_887 20200807 Sweden Oxel¨
osund 58.65613 17.11431
OL421194 Ulva lacinulata DK_115 20200721 Denmark Fredrikshavn_harbour 57.42541 10.52816
OL421394 Ulva lacinulata NO_132 20200709 Norway Farsund 58.06679 6.765637
OL421408 Ulva lacinulata SV_761 20200625 Sweden Båler¨
od 58.8917 11.2005
OL421299 Ulva linza DK_233 20200725 Denmark Bagenkop_1 54.75111 10.67343
OL421154 Ulva linza DK_073 20200716 Denmark Horslunde 54.95664 11.16232
OL421193 Ulva linza DK_114 20200721 Denmark Fredrikshavn_harbour 57.42541 10.52816
OL421320 Ulva linza NO_041 20200701 Norway Skjærhalden_1 59.02982 11.00613
OL421417 Ulva linza SV_773 20200625 Sweden Res¨
o_hamn 58.7999 11.1654
OL421465 Ulva linza SV_823 20200731 Sweden Haverdal_1 56.76606 12.6289
OL421111 Ulva prolifera DK_026 20200715 Denmark Gilleleje 56.12488 12.31448
OL421355 Ulva prolifera NO_086 20200703 Norway Nevlunghavn 58.96769 9.868426
OL421466 Ulva prolifera SV_824 20200731 Sweden Haverdal_1 56.76606 12.6289
OL421368 Ulva sp. 1 NO_101 20200708 Norway Brekkestø 58.19468 8.346613
OL421353 Ulva sp. 2 NO_084 20200703 Norway Nevlunghavn 58.96769 9.868426
OL421407 Ulva sp. 2 SV_759 20200625 Sweden Båler¨
od 58.8917 11.2005
OP267647 Ulva sp. 3 DK_018 20200715 Denmark Hundested_1 55.99406 11.90752
OP267679 Ulva sp. 3 NO_137 20200709 Norway Borshavn 58.10042 6.582716
OP267908 Ulva sp. 3 SV_403 20190627 Sweden G¨
oteborg I 57.70251 11.9247
OP268095 Ulva sp. 3 SV_717 20190726 Sweden Vilken 58.82587 11.03681
OL421264 Ulva sp. 3 DK_194 20200724 Denmark Gammel_Åbo 55.4683 9.680347
OL421469 Ulva sp. 3 SV_829 20200731 Sweden Haverdal_3 56.73906 12.63072
OP267895 Ulva sp. 4 SV_371 20190626 Sweden Daft¨
o camping 58.9011 11.1953
OP268048 Ulva sp. 5 SV_636 20190714 Sweden Hudiksvall 1 61.72538 17.11958
OP267689 Ulva sp. 5 SV_908 20200808 Sweden L¨
orudden 62.23017 17.6571
OL421206 Ulva sp. 6 DK_129 20200721 Denmark Hjørring 57.4763 9.794703
OL421252 Ulva sp. 6 DK_180 20200723 Denmark Ebeltoft 56.2275 10.62067
OL421263 Ulva sp. 6 DK_193 20200724 Denmark Gammel_Åbo 55.4683 9.680347
OP267862 Ulva sp. 6 SV_292 20190624 Sweden Fj¨
allbacka strand 58.59184 11.27837
OP267685 Ulva sp. 6 SV_779 20200625 Sweden Grebbestad 58.6836 11.258
OP267687 Ulva sp. 7 SV_841 20200801 Sweden H¨
ogan¨
as_2 56.20799 12.53921
OL421487 Ulva sp. 7 SV_848 20200805 Sweden Åhus 55.90788 14.30355
OL421280 Ulva sp. 8 DK_212 20200724 Denmark Sonderborg 54.90067 9.794234
OP267670 Ulva sp. 8 NO_051 20200701 Norway Gressvik_1 59.19525 10.79682
OP267677 Ulva sp. 8 NO_133 20200709 Norway Borshavn 58.10042 6.582716
OP267681 Ulva sp. 8 NO_143 20200709 Norway Nesvåg 58.33778 6.202459
OP267688 Ulva sp. 8 SV_896 20200807 Sweden Gr¨
add¨
o 59.76555 19.03097
MH538645 Ulva sp. 9 S_233 20140813 Germany Schilksee 54.4278 10.17172
OL421272 Ulva torta DK_203 20200724 Denmark Ørby_Hage 55.29254 9.66374
OL421377 Ulva torta NO_111 20200708 Norway Flekkerøy_2 58.07858 8.018074
OL421413 Ulva torta SV_768 20200625 Sweden Ross¨
o_Strand 58.84275 11.15182
S. Steinhagen et al.

Algal Research 73 (2023) 103132
6
The biodiversity strongly decreased across the Arkona Basin (6
taxonomically valid species, 1 unidentied species, species complexes,
singletons) and Bornholm Basin (4 taxonomically valid species, 1 un-
identied species, species complexes, singletons) whereas in the Baltic
proper only a reduced biodiversity (2 [plus 2 single drifting individuals
of U. compressa found at Kalmar, Sweden] taxonomically valid species, 2
unidentied species, species complexes, singletons) was observed
(Fig. 1). The lowest Ulva biodiversity in the Atlantic-Baltic Sea region
was observed in the Bothnian Sea (2 taxonomically valid species, 1
unidentied species, species complexes, singletons) and no Ulva spec-
imen was observed in the northern Bothnian Sea when salinities drop-
ped below 3 PSU (Fig. 1). Whereas most of the species detected in lower
salinities of the Baltic Sea were also found in fully marine ecosystems,
individuals of Ulva sp. 5 were only found in the Baltic proper.
In the whole sampling area 4 obligate foliose species (U. australis,
U. fenestrata, U. gigantea, U. lacinulata), 3 obligate tubular species
(U. exuosa, U. prolifera, U. torta), and 4 species with mixed morphol-
ogies (U. californica, U. compressa, U. intestinalis, U. linza) were found
among the taxonomically valid Ulva species. Notably, all observed un-
identied Ulva spp. (Ulva sp. 1 – Ulva sp. 9) were recorded with a tubular
morphology (Tables 2, 3).
3.3. Single species distribution patterns and ecotype appearances
Besides distinct distribution patterns of molecularly identied en-
tities (Fig. 1), specic ecotype appearances have been recorded within
this study. Especially ecotypes of the above-named Ulva species repre-
senting mixed morphologies show distinct appearances and range
margins within the Atlantic-Baltic Sea region (Table 2). For detailed
information on the single species and ecotypes see also the explanatory
texts in the supplementary content.
3.4. Species inventory: historical vs. recent species richness
For the green macroalgae present in the Atlantic-Baltic Sea region
different historic identication literature of macrophyte species have
been compiled. Within this study we directly compared our molecular
results with the most frequently applied identication keys and in-
ventory lists, including literature on the species rich areas of the NE
Atlantic [42] and the Swedish west coast [37,39,45], and the Baltic Sea
[37,38,40,41,44] (Table 3).
The present study implies that the expected historic species in-
ventory of the genus Ulva in the Atlantic-Baltic Sea region strongly di-
verges from our molecular based ndings (Table 3). The most frequently
used identication literature of the region lists 16 taxonomically
accepted Ulva species for the area, but we were able to molecularly
identify 11 Ulva species by respective peer-reviewed reference se-
quences (Table 3). Thereof, 8 coincided with historic listings, 3 were
observed within this and a recent study [53] for the rst time, and 8 Ulva
species were historically reported but have not been identied in our
large-scale biodiversity assessment (Table 3). For most of the eight
historically reported species, no reliable reference sequences were
available at GenBank.
With a total number of nine, the number of singletons and uniden-
tied sequences is <6 % (Supplementary Table S1) indicating that such
entities are rather uncommon which underlines the necessity of small-
scale sampling for biodiversity assessments within the genus Ulva.
Within this study we can conrm that mis-identication of Ulva spp.
probably led to diverging results among the molecular and historic
species inventory, it is however beyond the scope of the study to
determine if historically used species concepts are of validity.
4. Discussion
Our study points out that Ulva species diversity is still incomplete,
and that species and ecotype specic distribution patterns as well as
Table 2
Distribution in regard to salinity tolerance of the molecularly (tufA-based)
identied Ulva entities within the Atlantic-Baltic Sea gradient. Respective
salinity range margins of morphological ecotypes and ecotype specic habitat
distributions are listed. Footnotes highlight further relevant literature.
Species Salinity
range
[PSU]
Morphology Habitat
Ulva intestinalis 3–34 (often
in fresh
water
inows)
Tubular Supralittoral -
Infralittoral
Freshwater
inows
Rockpools
Attached
Epiphytic
5–12.3 Monostromatic
foliose (no
rhizoid)
a
Eulittoral -
infralittoral
Free-oating
Ulva linza 3–34 Tubular
b, c
Eulittoral –
infralittoral
Mainly
submersed
Attached
Epiphytic
8–34 Tubular base
foliose top
b, c
Eulittoral –
infralittoral
Mainly
submersed
Attached
Epiphytic
8–34 Foliose (no
rhizoid)
b
Eulittoral -
infralittoral
(rarely
supralittoral
when inated)
Free-oating
Ulva compressa >20–34 Tubular
d
Eulittoral –
infralittoral
Attached
Epiphytic
7.5–34 Foliose
d, e
Eulittoral -
infralittoral
Mainly free-
oating (rarely
attached)
Ulva prolifera 34–7.5 Tubular Eulittoral
Attached
Epiphytic
Ulva lacinulata 34–15.3 Foliose
f, g
Eulittoral
Attached
Free-oating
Ulva torta 34–15.3 Tubular Supralittoral -
infralittoral
Rockpools
Attached
Epiphytic
Ulva exuosa 34–16 Tubular Supralittoral -
infralittoral
Attached
Free-oating
Epiphytic
Ulva gigantea 34–16 Foliose Eulittoral
Attached
Epiphytic
Ulva fenestrata 34–20
(≈10)
Foliose
h
Eulittoral
Attached
Epiphytic
Ulva australis 34–25 Foliose Eulittoral
Attached
Epiphytic
Ulva californica 34–25 Tubular
i
Eulittoral
Attached
Epiphytic
34–25 Foliose
i
Eulittoral –
infralittoral
Free-oating
(continued on next page)
S. Steinhagen et al.

Algal Research 73 (2023) 103132
7
potentially invasive species distributions were widely mis-interpreted in
the Atlantic-Baltic Sea transect – even though the Baltic Sea is regarded
as the world's most intensely studied coastal area [65,66]. We conrm
this with three main lines of evidence: (1) The molecularly assessed
species inventory of our study strongly diverges from historic in-
ventories and those based on morphological identication of Ulva spp.
only and reveals not only a different number of present species but also
detects potentially invasive species in the region. (2) The distribution
patterns and range margins for Ulva spp. in the Atlantic-Baltic Sea region
differentiate strongly between the molecular results from this study and
historically stated distribution patterns. (3) Our study reveals the pres-
ence of several unidentied Ulva spp. in the area which could not clearly
be allocated to previously dened species concepts.
Considering the genus Ulva as being a central part of the growing
aquaculture industry in the area [13–16,74,75] and identifying the
marine aquaculture as an important vector for the dispersal of neophytes
and invasive species [9] detailed knowledge on the species specic
distributions is needed to support conservation efforts and foster the
preservation and restoration of our valuable marine ecosystems.
4.1. Molecular Ulva biodiversity of the Atlantic-Baltic Sea gradient
We identied 11 Ulva taxa and additionally at least nine unidentied
Ulva species and species complexes, or singletons in the Atlantic-Baltic
Sea transect. However, the 11 Ulva taxa can only be identied to taxo-
nomic species level with variable degrees of certainty. Only four entities
(U. australis, U. fenestrata, U. gigantea, U. lacinulata) could be assigned to
clades which included reference sequences from lecto- or holotype
material [50–52,54,73,90]. Although, our data reects a detailed pic-
ture of the current state of the molecular biodiversity of Ulva in the
Atlantic-Baltic Sea gradient the taxonomy of species and therefore the
correct taxonomic name application might change with the availability
of more sequenced Ulva type material and should therefore be carefully
checked. Whereas efforts in sequencing of mainly foliose Ulva species
have been made recently [50–52,54,73], many tubular species – despite
lately described ones (e.g. [25,46,49]) – lacking such utmost important
molecular investigations of type material. However, our study
underlines that sequencing of type material of tubular species is espe-
cially necessary to determine their taxonomic identity as many can have
variable and cryptic morphologies (e.g. [27,36,53,58,61]) and are
therefore often mistaken with other species. A good example within this
study is e.g. individuals clustering within the Ulva linza-complex.
Whereas the majority of individuals identied as U. linza clustered with
a reference sequence from Australia, Tasmania (JN029337), only few
individuals clustered with a reference sequence from California, USA
(KM254997). Both clades are closely related but clearly form single
clusters which are both supported with bootstrap support values >97. A
similar topology has been previously reported from phylogenetic ana-
lyses [53,61]. Notably, four homotypic and 25 heterotopic synonyms
were listed for U. linza [57] and sequencing of type material could vastly
contribute to disentangle the taxonomic obscurities within a complex
that has a wide distribution (e.g. [53,58,61,76,77]). These ndings
underline, that certain species of Ulva are truly cosmopolitan and being
distributed across a wide range of latitudes.
The necessity of molecular investigations of type material of tubular
Ulva species gets further supported by the fact, that all unidentied Ulva
spp., species complexes, and singletons of our study have been recorded
with a tubular morphology. To assess whether the unidentied Ulva
entities reect new or already described species – which might lack any
kind of molecular data – additional genetic markers need to be inves-
tigated, species delimitation methods should be applied, and respective
holo- and lectotypes of potential candidate species need to be visited.
Due to the prevailing COVID-19 situation that was not possible within
the present study and will therefore be treated in detail in an additional
study.
4.2. Species distribution
Similarly with the divergence between the recent molecular results
and the expected historic species inventory, also notable differences in
the distribution of species of historic studies and our molecular assess-
ment were made. In total 16 taxonomically valid species were histori-
cally mentioned for the area [37–42,44,45] and our data revealed the
presence of 8 of these species, whereas furthermore 3 molecularly
identied species have not been mentioned in species keys of inventory
lists of the region. This discrepancy among historic and recent ndings
gets even further supported by the presence of above named unidenti-
ed Ulva species, species complexes, or singletons. That the appearance
of cryptic and morphologically very variable species led to mis-
interpretations and false identications in the past has often been dis-
cussed in literature, but especially large-scale assessments, as the pre-
sent one, which has been conducted over >10,000 km of coastline and
over different salinity regimes (ranging from fully marine conditions to
nearly fresh-water state) can determine range margins of species. Such
species distributions and the revealing of range margins is on the one
hand side interesting from ecological perspectives as the species´studied
showed clear patterns of e.g. replacements of morphotypes of other
species which occupy similar niches under different salinity regimes, but
it is furthermore of importance for the emerging seaweed aquaculture
industry of sea-lettuces in the area [13,15,16,75]. The selection of
suitable Ulva strains depending on the location of farms and prevailing
abiotic conditions (e.g. salinity regimes) are important criteria for the
rapidly growing aquaculture sector [13,14,74,75].
Along with the decreasing salinity in the Atlantic-Baltic Sea region,
also the biodiversity of Ulva spp. decreased, as previously observed for
other marine and brackish species in the area [64]. It should however be
mentioned that the species biodiversity and distribution, reects the
status quo of the area and that annually and seasonally varying abiotic
conditions might move certain distribution margins. Furthermore,
future scenarios predict further desalinisation and changes in the water
biochemistry of the waterbodies in the Atlantic-Baltic Sea transect (e.g.
[64–66,78]) which will most probably affect species range margins and
the overall biodiversity. Therefore, constant monitorings, especially of
Table 2 (continued )
Species Salinity
range
[PSU]
Morphology Habitat
Ulva sp. 1 34–28 Tubular Eulittoral
Attached
Ulva sp. 2 [Ulva capillata] 34–14.4 Tubular
j
Supralittoral –
eulittoral
Attached
Epiphytic
Free-oating
Ulva sp. 3 34–14.4 Tubular Supralittoral –
eulittoral
Attached
Epiphytic
Free-oating
Ulva sp. 4 34–28 Tubular Eulittoral
Attached
Ulva sp. 5 5–6 Tubular Eulittoral
Attached
Ulva sp. 6 15–34 (6) Tubular Eulittoral
Attached
Ulva sp. 7 13.7–20 Tubular Eulittoral
Attached
Ulva sp. 8 15–34 Tubular Eulittoral
Attached
Ulva sp. 9 16.2 Tubular Eulittoral
Attached
a Blomster et al. [63]; b Steinhagen et al. [53]; c Steinhagen et al. [61];
d Steinhagen et al. [27]; e Steinhagen et al. [91]; f Hughey et al. [52]; g Fort et al.
[73]; h Hughey et al. [51]; i Steinhagen et al. [53]; j Steinhagen et al. [49].
S. Steinhagen et al.

Algal Research 73 (2023) 103132
8
invasive and nuisance species, should be emphasized. To gather species
specic information on their ecology, distribution, and molecular
salience, observed species of the Atlantic-Baltic Sea transect should be
discussed in detail:
4.3. U. intestinalis and U. linza
The species U. intestinalis and U. linza showed the widest distribution
and were present across salinities ranging from 36 to 3.5 PSU. They were
fastly decreasing in abundance after The Quark and mainly absent from
the Bothnian Bay, however we cannot say if that is due to the prevailing
low salinities <3PSU or other abiotic factors that e.g. inuence the
prevailing water chemistry [64–66]. If the adaptation of certain strains
of U. intestinalis and U. linza to fresh water conditions is therefore time-
limited needs to be investigated in detail.
Notably, when comparing our ndings to the literature all included
identication keys and inventories list U. intestinalis for the region and
most of them also U. linza [37–42,44,45]. Whereas U. intestinalis was
however a recognized species of the Baltic Sea, it seems that U. linza has
often been mis-identied in the past, as it is absent of species keys
focusing mainly on the Baltic proper e.g. Tolstoy et al. [44].
That U. intestinalis has a wide tolerance towards rapidly changing
temperatures and salinities, which include fresh water state, gets how-
ever underlined by the fact that rock-pools – which underlay strong
temperature and salinity uctuations depending on the atmospheric
condition – were mainly inhabited by individuals of U. intestinalis. The
adaptation of U. intestinalis and U. linza towards varying abiotic factors
and their appearance in disturbed ecosystems has been intensely
Table 3
Comparison of molecular (tufA)-based identication from the present study with inventory lists from the studied area (see footnote). Taxonomically valid species
names are marked in bold whereas hetero- and homotypic synonyms used in the investigated literature are listed below each species. The last two columns give a direct
comparison of today's taxonomically accepted Ulva spp. [57] of the investigated literature and the present molecular ndings.
Species name used in the literature [39] [41] [42] [45] [37] [44] [38] [40] Historical
presence
Molecularly
identied
Ulva australis Areschoug – – – – – – – – – (✓) ✓
Ulva laetevirens Areschoug – – – ✓* ✓ – – –
Ulva californica Wille – – – – – – – – – ✓
Ulva clathrata (Roth) C.Agardh – – – ✓ ✓ – – ✓ ✓ –
Enteromorpha clathrata (Roth) Greville ✓ ✓ ✓ ✓* – ✓ – –
Enteromorpha muscoides (Clemente) Cremades – – – ✓* – – – –
Enteromorpha ramulosa (Smith) Carmichael – ✓ ✓ ✓* – – – –
Ulva compressa Linnaeus – – – – ✓ – – ✓ ✓ ✓
Enteromorpha complanata Kützing ✓ – – – – – – –
Enteromorpha compressa (Linnaeus) Nees ✓ ✓ ✓ ✓ – ✓ – –
Ulva curvata (Kützing) De Toni – ✓ ✓ ✓ ✓ – – ✓ ✓ –
Ulva fenestrata Postels & Ruprecht – – – – – – – ✓ ✓ ✓
Ulva exuosa Wulfen – – – – ✓ – – ✓ ✓ ✓
Enteromorpha exuosa (Wulfen) J.Agardh – ✓ ✓ ✓ – ✓ – –
Enteromorpha biagellata Bliding ✓ – – – – – – –
Ulva gigantea (Kützing) Bliding – – – – – – – – – ✓
Ulva intestinalis Linnaeus – – – – ✓ – ✓ ✓ ✓ ✓
Enteromorpha intestinalis (Linnaeus) Nees ✓ ✓ ✓ ✓ – ✓ – –
Ulva intestinaloides (Koeman & Hoek) H.S.Hayden, Blomster, Maggs,
P.C.Silva, Stanhope & Waaland
– – – – – – – ✓
Ulva kylinii (Bliding) H.S.Hayden, Blomster, Maggs, P.C.Silva,
Stanhope & Waaland
– – – ✓ ✓ – – – ✓ –
Enteromorpha kylinii Bliding ✓ – ✓ – – – – –
Ulva lacinulata (Kützing) Wittrock – – – – – – – – ✓ ✓
Ulva scandinavica Bliding – – ✓ ✓* ✓ – – –
Ulva lactuca Linnaeus ✓ ✓ ✓ ✓ ✓ ✓ ✓ – ✓ –
Ulva rotundata Bliding – – ✓ – – – – –
Ulva linza Linnaeus – – – – ✓ – ✓ ✓ ✓ ✓
Enteromorpha ahlneriana Bliding ✓ ✓ ✓ ✓* – – – –
Enteromorpha linza (Linnaeus) J.Agardh ✓ ✓ ✓ ✓ – – – –
Ulva paradoxa C.Agardh – – – – – – – ✓ ✓ –
Ulva pilifera (Kützing) ˇ
Skaloud & Leliaert – – – – – – – – ✓ –
Enteromorpha exuosa subsp. pilifera (Kützing) Bliding – – – ✓ – – – –
Ulva prolifera O.F.Müller – – – – ✓ ✓ ✓ ✓ ✓ ✓
Enteromorpha procera K. Ahlner – – – ✓* – – – –
Ulva procera (K.Ahlner) H.S.Hayden, Blomster, Maggs, P.C.Silva,
Stanhope & Waaland
– – – – ✓ ✓ ✓ ✓
Enteromorpha prolifera O.F.Müller (J. Agardh) ✓ ✓ ✓ ✓ – – – –
Enteromorpha simplex (K.L.Vinogradova) R.P.T.Koeman & Hoek – – – ✓ – – – –
Ulva simplex (K.L.Vinogradova) H.S.Hayden, Blomster, Maggs, P.C.
Silva, Stanhope & Waaland
– – – – ✓ – – ✓
Ulva radiata (J.Agardh) H.S.Hayden, Blomster, Maggs, P.C.Silva,
Stanhope & Waaland
– – – – – – – – ✓ –
Enteromorpha radiata J.Agardh – ✓ – – – – – –
Ulva rigida C.Agardh – ✓ ✓ ✓ ✓ – – – ✓ –
Ulva torta (Mertens) Trevisan – – – ✓ ✓ – – ✓ ✓ ✓
Enteromorpha torta (Mertens) Reinbold – ✓ – ✓ – – – –
The reference literature and species inventories listed above are focusing on macrophytes of the whole Baltic Sea [41] and the Swedish Baltic coast [44], the whole
Swedish coastline [37,38] and the Swedish west coast [39], [45], the Norwegian Atlantic coast and parts of the Skagerrak Rueness [42], as well as on the whole Danish
coastline [40]. Asterisks indicate notes on taxonomic changes in the 2010 update of the University of Gothenburg (UoG) identication key (1988).
S. Steinhagen et al.

Algal Research 73 (2023) 103132
9
discussed in previous studies (e.g. [61,63,79]). With their wide distri-
bution, tolerance towards changing environmental conditions, and
interesting biochemical contents [80], U. intestinalis was evaluated as a
suitable species for aquaculture purposes in the wider Baltic Sea region
[75,80]. Since U. linza exhibits a wide distribution in the Atlantic-Baltic
Sea transect and has been previously evaluated as a putatively good
candidate in Integrated Multitrophic Aquaculture settings [81], its
application for the Baltic aquaculture industry should be investigated in
detail. Another notable point is the aberrant foliose morphotype of
certain individuals of U. intestinalis observed in the Baltic Sea. Foliose,
monostromatic fronds of U. intestinalis have been observed before along
the Finish coast [63] and were also observed in our study along the
Swedish, Finish and German coasts where they were often involved in
mass-accumulations. The appearance of morphologically overlapping
foliose Ulva species and/or ecotypes which lead to mis-identications in
the past will be discussed in detail below.
4.4. U. compressa and U. prolifera
U. compressa and U. prolifera were abundantly present until the
Bornholm Basin and were found in salinities ranging from 36 to 7.5 PSU.
All of the investigated literature lists U. prolifera (or its homo- and het-
erotypic synonyms) for the Atlantic-Baltic Sea area and therefore our
ndings are widely in accordance with previous ndings for the region.
It should however be mentioned, that difference in the historically ex-
pected and molecularly veried distribution of U. prolifera were
encountered. Whereas, identication keys focussing on the Baltic Sea
only listed U. prolifera (or its respective synonymized species) as present
in the Baltic Sea proper [41,44], this cannot be conrmed by our nd-
ings. Since the Ulva species diversity was found to be very reduced in the
Baltic Sea, it is highly likely that present species of the Baltic Sea region
develop aberrant morphotypes (see also [61,63]) which could have been
mis-interpreted as U. prolifera in the past, due to applying morphological
identication criteria only. Especially species such as Ulva procera which
got later synonymized with U. prolifera [57] were described to be
branched and have been historically recorded for the Baltic Sea area
[44]. It is therefore highly likely that branched individuals of
U. intestinalis and/or U. linza have been mistaken with other species in
the past within the Baltic Sea region. That individuals of U. intestinalis
and U. linza are indeed able of developing branched morphotypes under
the varying salinity regimes of the region has been shown before [61].
Instead, the tubular individuals of U. compressa have only been
observed in marine environments and were never found below salinities
of <20.5 PSU. However, after the Danish straits until the Bornholm
Basin distromatic and foliose individuals of U. compressa have been
found which mainly appeared drifting and which in some occasions
were involved in mass-accumulations. These results are in accordance
with previous ndings [27,53,82]. However, striking discrepancies
among the molecularly assessed distribution of U. compressa with the
morphological identication criteria of the literature were made.
Whereas some species keys list the tubular individuals of U. compressa to
be abundantly present until the uppermost parts of the Bothnian Sea
[44] other identication keys do not list U. compressa at all for the area
[38]. This underlines the overlapping morphology of Ulva species in the
area which evidently led to mis-interpretations in the past.
4.5. U. lacinulata and U. torta
The same distribution area ranging from the Atlantic until the
Arkona Basin was shared by individuals of U. lacinulata and U. torta and
individuals of these species were found in salinities ranging from 36 to
15.3 PSU.
More recently, type material of U. lacinulata has been molecularly
investigated [52] which conrmed the mis-application of names among
individuals of Ulva rigida and U. lacinulata and our genetic investigation
evidently conrmed that U. rigida was absent from the investigated
region but that U. lacinulata is a relatively frequent species (see also
[27,53]). When comparing recent ndings with the literature it becomes
obvious that the with U. lacinulata synonymized species Ulva scandi-
navica has been listed for the higher salinity areas such as the Atlantic
and Skagerrak region [37,42,45] but never for the geographic region
among the Kattegat and Arkona Basin [38,40,44], where it was detected
within the present study.
The tubular species U. torta has been correctly listed by several
identication keys and species lists [37,40,41,45] and it should be noted
that U. torta was frequently found as inhabitant of rock pools - besides
the dominant stands of U. intestinalis – and although the salinity within
these ecosystems uctuates, individuals of U. torta have not been found
in the Baltic proper within our study.
4.6. U. gigantea and U. exuosa
Individuals of U. gigantea and U. exuosa were found between the
fully marine waterbodies of the Atlantic and the brackish waterbody of
the Kiel bay (16 PSU). It should however be mentioned that both species
were rather infrequently found and less abundant than other Ulva spe-
cies in the area. That the distribution limit of the species is within the
Kiel bay could also be inuenced by the Kiel Canal which is one of the
most travelled articial waterways worldwide and directly connects the
fully marine German North Sea with the brackish Baltic Sea [61]. It is
known that ships can function as vector for marine species dispersal [83]
and since both of the species were only found in higher salinity areas and
exclusively within the vicinity of the Kiel Canal (see also [53]), it could
have been possible that single individuals, adapted to lower salinity
regimes and established in the area.
U. gigantea is considered an invasive species within the area and has
therefore not been listed in any of the species keys or inventory lists of
the area before and was rstly identied by Steinhagen et al. [53] along
the German coasts.
Although, we did not observe individuals of U. gigantea associated
with mass-accumulations or green tides within the present study, pre-
vious studies on U. gigantea strains of the Atlantic conrmed its prolif-
erating character [84]. Therefore, the expansion and distribution of
U. gigantea in the Atlantic-Baltic Sea transect should be carefully
monitored in the future.
Nearly all literature of the region lists U. exuosa as an abundant
species [37,39–42,44,45] which stands in strong contradiction to our
ndings. It has furthermore been discussed by several authors, that
U. exuosa is a frequent species within fresh water environments
[85,86], we were however not able to verify this species for the wider
Baltic Sea region. Although, the sampling was conducted very thorough,
it is however possible that the ne and delicate thalli have been over-
looked at some sites. To determine the distribution limit of inconspic-
uous and delicate species the approach of eDNA metabarcoding would
be an optimal solution [87,88]. Another explanation for the strong
discrepancies among our study and existing literature of the region
could be, that unidentied Ulva spp. of the region share a similar
morphology with individuals of U. exuosa (data not shown). The po-
tential of mis-interpretations of U. exuosa with undescribed species or
species complexes is therefore mandatory to investigate, in order to
disentangle historic confusions.
4.7. U. fenestrata (previously regarded as U. lactuca)
The most striking discrepancies among historic and recent distribu-
tion patterns of Ulva species in the Atlantic-Baltic Sea gradient were
found for U. lactuca. Until Nielsen and Lundsteen's [40] description of
the Danish algae ora which did not list U. lactuca, all historic literature
until then has cited U. lactuca as being abundantly present throughout
the Atlantic and Skagerrak [38,39,42], Kattegat [38,39], Kiel bight
[38,41], Arkona Basin [38,41,44], and vast parts of the Baltic Sea
[38,44].
S. Steinhagen et al.

Algal Research 73 (2023) 103132
10
Hughey et al. [51] revealed the ongoing confusion of U. lactuca with
other Ulva species by molecular investigations of the type specimen of
U. lactuca, and evidently conrmed, that U. lactuca is a tropical species,
whereas northern hemisphere individuals were molecularly identied as
U. fenestrata. Here, we provide a completely revised picture of the dis-
tribution of foliose Ulva species and morphotypes in the Atlantic-Baltic
Sea region, which were mistaken by their overlapping morphology
with that of U. lactuca and therefore remained undetected and mis-
identied for so long. Our study revealed that several foliose Ulva spe-
cies (e.g. U. australis, U. fenestrata, U. gigantea, U. lacinulata) or ecotypes
of species (e.g. U. compressa, U. intestinalis, U. linza) can be found in the
investigated area, however U. fenestrata was restricted to relatively high-
salinity habitats, >17.2 PSU (except for individuals collected in the
vicinity of Norway's largest river Glomma [10 PSU] which had the main
discharge during the sample period and therefore probably lower
salinity levels are reected) and absent after the Danish straits. These
ndings coincide with rst studies carried out along the German
coastline [27,53]. Notably, within the Baltic proper we only found
foliose ecotypes of one Ulva species and these were aberrant morpho-
types of U. intestinalis, which have been previously recorded from green
tides at the Finish coasts [63]. Such foliose individuals of U. intestinalis
were never found in salinities above 12.3 PSU and they were absent from
the Atlantic, Skagerrak, Kattegat, and Kiel bay. As all of the investigated
individuals were found drifting and lacking any rhizoidal zone, they
might be adapted to a free oating life-history which has been recorded
for the Baltic Sea before [63].
The as invasive regarded species U. australis and U. californica have
been limited in their distribution to the higher salinity waterbodies of
the Atlantic and Skagerrak and have been found infrequently. Both
species have been recorded by Steinhagen et al. [53] at the German
coasts but were never mentioned before in the identication literature of
the area. It should however be mentioned that the species key of the
University of Gothenburg [45] as well as the inventory list Dyntaxa [37]
list U. australis´synonym, Ulva laetevirens, for the area. Since U. laetevirens
has been previously confused with U. rigida and therefore with
U. lacinulata [52], these database listing should be regarded with
caution (see also Table 3). Although U. australis is known to be
responsible for green tides in the Atlantic (e.g. [50]) we have not
observed any mass-accumulations of this species in the investigated
region. The distribution and abundance of both, U. australis and
U. californica should however be carefully monitored in the future.
Our study underlines, that intense samplings with relatively small
raster pitches are needed in order to encounter rare ecotype forms such
as drifting ecotypes (e.g. foliose individuals of U. compressa or
U. intestinalis), and to ll gaps within the still unresolved Ulva biodi-
versity. Within the 1000 individuals examined only 59 sequences have
been encountered, which belonged to unidentied species, unidentied
species complexes, or unidentied singletons. Individuals with uniden-
tiable sequences were found throughout the whole study region but
were predominantly present in higher salinity waters.
Especially within the EU efforts were made to molecularly identify
foliose Ulva species [27,50–52,73] as some of them possess a certain risk
of forming green tides and negatively affect coastal ecosystems
[16,53,89], but furthermore some native foliose species are regarded
important crop species in the emerging seaweed aquaculture markets
[11,13–16]. However, latest results on biodiversity assessments within
the genus Ulva revealed, that especially among the tubular Ulva species
strong taxonomic discrepancies prevail [25,53] and that taxonomic and
systematic efforts in these groups are required. This gets reected by the
present study as well as all of the unidentied Ulva entities exhibited a
tubular morphology.
4.8. Unidentied Ulva spp.
Whereas individuals of the Ulva sp. 2 cluster and those dened as
species complex Ulva sp. 3 had a clear distribution which stretched from
the marine waters of the Atlantic until the Arkona Bay (14 PSU), in-
dividuals of the species complexes Ulva sp. 6 and Ulva sp. 8 were
recorded from the marine Atlantic until the Kiel Bay (PSU 15.6),
whereas a single population of species complex Ulva sp. 8 was found in
the wider Stockholm archipelago (5.4 PSU). Individuals which belonged
to the clusters Ulva sp. 1, 4, 5, 7, 9 showed no gradual distribution and
therefore no distribution margins could be dened. It should however be
mentioned that individuals of Ulva sp. 5 were only found in the low
salinity waters of the Bothnian Sea. However, cluster Ulva sp. 2 has
recently been describes as the species Ulva capillata Steinhagen [15,16].
It however requires an intense collection of more individuals of the
other unidentied species, species-complexes, and singletons, in order
to designate their true taxonomic identity, evaluate their ecology, and
assess their distribution.
When summarizing the molecular data of the present study and
comparing it to the historic species inventories and keys it becomes
obvious that based on morphological identication criteria more species
have been listed for the Baltic Sea region than have actually been
molecularly validated. This on the one hand side supports the necessity
of molecular species identication within the genus Ulva but further-
more evidently conrms the high morphological variability of the actual
Ulva species and ecotypes that can be found in the Atlantic- Baltic Sea
transect. This offers a perfect study area for testing species hypothesis, or
answering evolutionary questions, but our study also enables future
assessments of ecologically and genetically driven range margins of
species or ecotypes within the genus Ulva.
Whereas the most striking results were obtained for Ulva species
detected in the area, it should not remain unmentioned, that several
taxonomically valid species, which have been previously listed for the
investigated area have not been validated within this study by molecular
methods, namely: Ulva clathrata, Ulva curvata, Ulva kylinii, Ulva lactuca,
Ulva paradoxa, Ulva pilifera, Ulva radiata, Ulva rigida. Although, we
focused on different ecosystems and habitats and included with 287
sampling sites a close-meshed sampling design, it could have been
possible that rare species could have remained undetected. To exclude
such bias of undetected rare species in DNA barcoding surveys, an
approach including eDNA sampling would be benecial. Furthermore,
since sequenced type specimens for many of the Ulva species are
missing, we cannot exclude, that some of the unidentiable sequences
observed in our study could reect rst sequences of the above named
undetected species. However, due to the absence of molecular reference
material for some of the undetected species it is necessary to include
morphological observations and investigations of historical type
vouchers. This is especially necessary for species such as U. clathrata,
U. curvata, and U. kylinii which all have their type locality in the wider
Baltic Sea region [57].
5. Conclusion
The current morphological concepts that were historically used for
the identication of Ulva species and their respective distribution and
range margins in the Atlantic-Baltic Sea transect were neither in
agreement with the species inventories and identication keys of the
area nor with the actual morphology of species that are present. We here
provide a completely revised picture of the status quo of the molecular
biodiversity of Ulva spp. within the Atlantic-Baltic Sea transect and
dene distribution patterns and range margins for the different species
as well as unravel the existence of multiple invasive and unidentied
species of the investigated area. Therefore, the observations of the pre-
sent study provide a basis for e.g. the development of managing efforts,
invasive species tracking, as well as for strain selection of the emerging
seaweed aquaculture industry.
CRediT authorship contribution statement
SST: conceptualization of the study, implementation of study,
S. Steinhagen et al.

Algal Research 73 (2023) 103132
11
investigation, data analyses, visualization, and original draft. SH: data
collection, eld work, analyses and rening of draft. GT: funding
acquisition, eld work, and rening of draft. HP: funding acquisition
and rening of draft. All authors: read and agreed to the published
version of the manuscript.
Declaration of competing interest
The authors declare no competing interests. Further the authors
report no commercial or proprietary interest in any product or concept
discussed in this article.
Data availability
The tufA sequences generated in this study to genetically identify
Ulva species are deposited in GenBank and all accession numbers
including prevailing environmental parameters of the respective sam-
pling sites can be found in Table S1. The original contributions pre-
sented in this study are included in the article/supplementary materials,
further inquiries can be directed to the corresponding author/s.
Acknowledgements
The authors thank the Formas-funded ‘A manual for the use of sus-
tainable marine resources’ project (Grant no. 2022-00331) for nancial
support. Furthermore, this project received nancial support from the
ULTFARMS project funded by the European Union (Grant no.
101093888). We would like to thank Marlene Jahnke, Annelous Oer-
bekke, Gunnar Cervin, Alexandra Kinnby, Florian Weinberger, Christian
Pansch, Henna Rinne, Sonja Salovius-Lauren, Elina Leskinen, and Jaa-
nika Blomster for the contribution of additional samples. Furthermore,
we want to thank Jeanette Ågren and Louise Kram´
ar for their help
during lab-work and morphological analyses.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.algal.2023.103132.
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