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Citation: Irvine, T.; Wysor, B.;
Beauvais, A. A Molecular-Informed
Species Inventory of the Order
Ceramiales (Rhodophyta) in the
Narragansett Bay Area (Rhode Island
and Massachusetts), USA. Diversity
2024,16, 554. https://doi.org/
10.3390/d16090554
Academic Editor: Stephan Koblmüller
Received: 29 June 2024
Revised: 5 August 2024
Accepted: 26 August 2024
Published: 5 September 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
diversity
Article
A Molecular-Informed Species Inventory of the Order
Ceramiales (Rhodophyta) in the Narragansett Bay Area
(Rhode Island and Massachusetts), USA
Thomas Irvine 1, 2, *, Brian Wysor 2,* and Alicia Beauvais 2
1
School of Life Sciences, University of Hawai‘i at M
¯
anoa, 3190 Maile Way Room 101, Honolulu, HI 96822, USA
2Department of Biology, Marine Biology and Environmental Science, Roger Williams University,
Bristol, RI 02809, USA
*Correspondence: tirv41@hawaii.edu (T.I.); bwysor@rwu.edu (B.W.)
Abstract: Narragansett Bay is an estuarine system in the western North Atlantic Ocean that harbors a
diverse marine flora, providing structure, habitat, and food for native biodiversity. This area has been
the center of numerous environmental, biological, ecological, and oceanographic studies; however,
marine macroalgae have not been extensively examined using modern molecular methods. Here, we
document the biodiversity of the red algal order Ceramiales based on DNA sequence comparisons of
the 3
′
end of the RuBisCo large subunit (rbcL-3P) and the universal plastid amplicon (UPA). Thirty-
seven distinct species of this order were identified and validated with molecular data, including
five new species reports and at least one new report of an introduced species, Antithamnionella
spirographidis, in the vicinity of Narraganset Bay. Novel sequence data were generated for numerous
species, and it was discovered that the UPA marker, which has been less frequently used in red
algal floristics, revealed an identical inventory of ceramialean algae as the rbcL-3P marker. Thus,
the shorter length of the UPA marker holds promise for DNA metabarcoding studies that seek to
elucidate biodiversity across algal phyla.
Keywords: algal floristics; Anthithamnionella spirographidis; DNA barcoding; molecular-assisted
identification; Narragansett Bay; seaweed biodiversity
1. Introduction
Introduced species are organisms that successfully establish a new population outside
of their natural range, frequently as a result of human activity. These organisms are of
particular importance because of their potential to wreak havoc on the ecosystems into
which they are introduced [
1
,
2
]. Algae introduced to marine and estuarine ecosystems can
cause declines in biodiversity, potentially creating more uniform environments that are
more vulnerable to environmental disturbances and climate change [
3
,
4
]. Furthermore,
preserving biodiversity in marine ecosystems is integral to maintaining ecosystem services,
such as productive fisheries, ecotourism, and shoreline protection [
5
]. Introduced algae
can also reshape ecosystems by outcompeting sessile invertebrates for space to grow [
6
]
and by replacing primary producers [
7
], both of which cause declines in animals that feed
exclusively on these native producers [8,9].
Marine algae can be introduced to new habitats through a variety of vectors, including
ballast water from ships [
10
], growing on ship hulls [
11
], intentional or accidental release
into a non-native ecosystem [
12
], or through poleward range expansions [
13
]. Poleward
range expansions of marine algal species are also becoming increasingly common due
to climate change, which increases sea surface temperatures, driving algal species into
cooler habitats that were previously uninhabitable for tropical and subtropical species. This
can be problematic as more equatorial populations begin to shift poleward and replace
Diversity 2024,16, 554. https://doi.org/10.3390/d16090554 https://www.mdpi.com/journal/diversity
Diversity 2024,16, 554 2 of 42
colder-water species, causing breakdowns in local ecosystems that rely on the presence of
the native colder-water species [14].
One particularly notable group of algae that has a greater propensity for invading
or being introduced to new habitats than any other group of algae due to its ability to
foul shipping hulls and its general resilience when introduced to new habitats is the
order Ceramiales [
15
]. The order Ceramiales (phylum: Rhodophyta) is a highly diverse
group of red algae encompassing over 2700 species. This group is the most species-rich
group of rhodophytes, making up almost 40% of all described red algal species [
16
].
Algae within this order are morphologically diverse, exhibiting filamentous, foliose, and
fleshy morphotypes with a wide variety of branching patterns, cell morphologies, and
reproduction strategies [
17
]. Furthermore, recent studies examining the Ceramiales have
found many new and cryptogenic species that were previously overlooked or mistakenly
lumped under names with other algae species [
18
–
20
]. High intraspecies morphological
plasticity, along with morphological convergence, has made algal classification challenging,
especially within the order Ceramiales [21,22].
Due to the challenges of classifying Ceramiales, molecular techniques have become
the standard for reconciling morphological identifications and classifying red algae. DNA
barcoding provides an objective method by which algal diversity can be scrutinized despite
well-known problems of morphological convergence and morphological plasticity that
make it challenging to identify species by morphology alone [
23
]. DNA barcoding is a
method used for identifying species based on the divergence of sequences of a particular
gene between individuals. This method has been used extensively across the world to
assess algal diversity to resolve taxonomic uncertainties of easily confused red algal species,
revealing cryptic diversity and objectively determining what species are present in a certain
ecosystem [
21
,
22
,
24
]. A few examples of large-scale DNA barcoding studies include work
on Rhodophyta of the Hawaiian archipelago [
25
], turf algae in the Pacific Panama [
26
], and
invasive/introduced species in the Azores [
27
], among many others. While specimen-by-
specimen studies, such as the ones mentioned above, effectively assess algal biodiversity,
they are limited by incomplete libraries of publicly available sequence data, which can lead
to erroneous identifications [
28
]. Incomplete public databases also restrict the capabilities
of metabarcoding studies that attempt to identify species assemblages and the biodiversity
of entire environments [
29
]. Hence, there is an exigence for more specimen-by-specimen
studies to generate reliable sequence data for this group of organisms, so that specimen-
independent studies have a reliable foundation that they can draw upon.
In this study, sequences from two different chloroplast-encoded genes were used: Do-
main V of the plastid-encoded large ribosomal subunit gene, also known as the universal
plastid amplicon (UPA) [
30
], and the 3
′
end of the RuBisCO large subunit gene (rbcL-3P) [
31
].
The rbcL-3P marker is a widely used marker for phylogeny and identification of marine algae,
as it is conserved between members of the same species but diverges between genera. Public
sequence repositories are replete with rbcL sequence data, which makes this marker partic-
ularly useful for contextualizing newly generated sequence data. A potential disadvantage,
though, is that the rbcL-3P marker can diverge at priming sites, such that different lineages
may require different primer sets to amplify effectively [
32
]. The UPA marker amplifies across
nearly the full range of autotrophic life with only a single set of primers, and its short length
(~370 bp) requires fewer sequence reads than the full rbcL gene, which can reduce overall costs.
Furthermore, the short length of UPA suits it well to next-generation sequencing strategies
because its full length can be recovered. Despite these advantages, the UPA marker has not
been widely used, and thus public sequence repositories remain incomplete. Where it has
been used for floristic treatments, estimates of species richness are largely concordant with
those determined from the rbcL-3P and COI-5P sequences [
25
,
26
]. Since we have enjoyed
greater success amplifying the rbcL-3P marker over the COI-5P marker, we used it as the com-
parative foundation to establish a preliminary UPA context for future, specimen-independent
metabarcoding endeavors on a taxonomically challenging group in a well-studied estuary,
subject to the pressures of human disturbance, including global climate change. Here, we
Diversity 2024,16, 554 3 of 42
compare contemporary, molecularly-validated marine flora with historical reports from the
Narragansett Bay Area (NBA). Using DNA barcoding, morphological analysis, and multiple
algal reference compendia, 37 species of the order Ceramiales were identified in the NBA,
including an introduced species not previously recorded in the western Atlantic. Sequence
data generated in this study establish a molecular foundation tied directly to contemporary
specimen vouchers and morphological species determinations. This foundation provides a
baseline for future study of introduced species in the NBA, and considering that the order
Ceramiales includes numerous introduced species, and at least one new introduced species is
reported here, there is a need for such a baseline study [
33
]. Furthermore, this foundation is
necessary for future monitoring projects that would rely on specimen-independent molecular
techniques, including studies that seek to explore changes in biodiversity or ecosystem niche
shifts as a result of climate change or other disturbances.
2. Materials and Methods
2.1. Specimen Collection
The majority of sequenced specimens were collected over the summer and fall of
2023. Ten sites representing a variety of intertidal and shallow subtidal habitats from
around the NBA were visited over the course of the summer and fall from the end of May
to early November 2023 (Figure 1). Algal specimens were collected while snorkeling or
wading. In situ images as well as lab macroscopic and microscopic images were recorded
for newly collected specimens. New specimens were also entered into the Marine Algae
of Rhode Island (MARI) database maintained in the Seaweed Biodiversity Lab at Roger
Williams University, and both herbarium presses and silica gel vouchers were created and
saved in RWU holdings. Many specimens collected in the summer of 2022, as well as older
herbarium specimens and silica gel vouchers from the NBA, were also sequenced to include
species that were not collected over the summer of 2023.
Diversity 2024, 16, x FOR PEER REVIEW 4 of 43
Figure 1. Map of collection site locations (red dots) in the Narraganse Bay Area for fresh specimen
collection in 2023. The dark gray box in the inset shows the main map with respect to a slightly more
removed collection site, Lighthouse Beach in Chatham, MA.
2.3. DNA Extraction, Amplification and Sequencing
Extraction of total genomic DNA was completed using the BioLine MyTaq Extract-
PCR Kit (Meridian Bioscience; Cincinai, OH, USA) for almost all samples collected after
the spring of 2023, including live, silica-gel-dried, and herbarium vouchers. Extractions
from silica-gel-dried vouchers completed before spring 2023 were performed with the
DNEasy Plant Mini-Kit (QIAGEN Sciences Inc.; Germantown, MD, USA). For the MyTaq
Extract-PCR Kit extraction method on live, fresh specimens, about 5 mm of algal tissue
from a new branch tip was added to the extraction solution and incubated overnight at
75 °C. The samples were then centrifuged at 14 krpm for two minutes to pellet excess
undissolved tissue. About 85 µL of the supernatant, the isolated total genomic DNA, was
retained. The optimal incubation time appears to be around 22 h, although clean se-
quences were obtained from extracts incubated from as short as 13 h to as long as 24 h.
Following extraction, 1:10 dilutions of the DNA extracts were prepared to be used in PCR,
as preliminary experiments showed that diluted extracts were far more likely to amplify
than undiluted extracts, and these samples often amplified much stronger than the undi-
luted extracts if both samples amplified. The primers used for both the rbcL-3P and UPA
loci are given in Table 1.
Table 1. Primer names and sequences used in this study.
Marker Name Sequence (5′-3′) Source
UPA p23SrV_f1 GGA CAG AAA GAC CCT ATG AA [25]
Figure 1. Map of collection site locations (red dots) in the Narragansett Bay Area for fresh specimen
collection in 2023. The dark gray box in the inset shows the main map with respect to a slightly more
removed collection site, Lighthouse Beach in Chatham, MA.
Diversity 2024,16, 554 4 of 42
2.2. Morphological Examination
All specimens were examined morphologically and compared to historical NBA re-
ports from four main compendia, including Sears [
34
], Villalard
−
Bohnsack [
35
], Mathieson
& Dawes [
17
], and Saunders [
36
]. For species where there were discrepancies with historical
reports, further literature review was conducted. Commonly referenced macroscopic characters
for separating algae of the order Ceramiales include general thallus arrangement and branching
patterns, while microscopic characters include seriation and cortical development on main axes.
2.3. DNA Extraction, Amplification and Sequencing
Extraction of total genomic DNA was completed using the BioLine MyTaq Extract-
PCR Kit (Meridian Bioscience; Cincinatti, OH, USA) for almost all samples collected after
the spring of 2023, including live, silica-gel-dried, and herbarium vouchers. Extractions
from silica-gel-dried vouchers completed before spring 2023 were performed with the
DNEasy Plant Mini-Kit (QIAGEN Sciences Inc.; Germantown, MD, USA). For the MyTaq
Extract-PCR Kit extraction method on live, fresh specimens, about 5 mm of algal tissue from
a new branch tip was added to the extraction solution and incubated overnight at 75
◦
C.
The samples were then centrifuged at 14 krpm for two minutes to pellet excess undissolved
tissue. About 85
µ
L of the supernatant, the isolated total genomic DNA, was retained. The
optimal incubation time appears to be around 22 h, although clean sequences were obtained
from extracts incubated from as short as 13 h to as long as 24 h. Following extraction, 1:10
dilutions of the DNA extracts were prepared to be used in PCR, as preliminary experiments
showed that diluted extracts were far more likely to amplify than undiluted extracts, and
these samples often amplified much stronger than the undiluted extracts if both samples
amplified. The primers used for both the rbcL-3P and UPA loci are given in Table 1.
Table 1. Primer names and sequences used in this study.
Marker Name Sequence (5′-3′) Source
UPA p23SrV_f1 GGA CAG AAA GAC CCT ATG AA [25]
p23SrV_r1 TCA GCC TGT TAT CCC TAG AG [25]
rbcL-3P F753 GGA AGA TAT GTA TGA AAG AGC [37]
rbcLrevNEW ACA TTT GCT GTT GGA GTY TC [32]
R1442
AAA CAT TAG CTG TTG GAG TTT CTA C
[38]
Standard PCR reagents (Table 2) and thermocycling profiles (Table 3) were used for amplifying the target
gene regions.
Table 2. Individual reaction ingredients for the 20
µ
L PCR experiments. The same reagents and
volumes were used to create PCR cocktails for both target genes using the primers listed in Table 1.
Reagent Reaction Volume (mL) Final PCR Concentration
BioLine 2×MyTaq Red HS Mix 10 1×
10 mM Forward Primer 1 0.5 mM
10 mM Reverse Primer 1 0.5 mM
5M Betaine 4 1 M
PCR Water 3 --
DNA Template 1 --
Table 3. Thermocycling profile for PCR. The same parameters were used for both markers and are
shown below.
PCR Stage Temperature (◦C) Time (min) # Cycles
Initial Denature 95 5 1
Cycle Start Denature 95 0.5 35
Annealing 50 0.5 35
Cycle End Extension 72 1.5 35
Final Extension 72 5 1
Diversity 2024,16, 554 5 of 42
Following amplification, PCR products were electrophoresed on 1.2% UltraPure LMP
agarose (Invitrogen; Carlsbad, CA, USA) gels prepared with a final concentration of 0.5x
GelGreen Nucleic Acid Stain (Biotium; Fremont, CA, USA). Amplicons were excised from
the gel with a clean razor blade, and DNA was isolated using the Bioline Isolate II PCR
and Gel Kit (Meridian Bioscience; Cincinatti, OH, USA) following standard protocol, but
substituting PCR water for the provided elution reagent. Two microliters of each cleaned
PCR product was then immediately used to determine DNA concentrations (ngDNA/
µ
L)
using the NanoDrop One C spectrophotometer (Thermo Scientific; Waltham, MA, USA).
All cleaned PCR amplicons were prepared for Sanger sequencing at the University of
Rhode Island Molecular Informatics Core Facility, using approximately 5 and 8.75 ng
templates for UPA and rbcL-3P, respectively, and 5 pmol/reaction of forward or reverse
primer. The Applied Biosystems BigDye
®
Terminator v3.1 chemistry was used, and cycle
sequencing products were analyzed on the ABI 3500xl genetic analyzer following removal
of unincorporated reagents using magnetic bead clean-up.
2.4. Molecular Analysis
DNA sequences were analyzed using Geneious Prime software (Version 2022.1.1,
Dotmatics, Auckland, New Zealand). Completed sequences were trimmed to remove the
primer sequences and standardized sequence lengths for analysis. The rbcL-3P sequences
were 667–670 base pairs in length, and the UPA sequences ranged from 368 to 370 base
pairs. Two types of phylogenetic trees were generated using different algorithms. Prior
to generating trees for both algorithms, the sequences were aligned using MUSCLE [
39
]
through Geneious Prime. From the MUSCLE alignment, UPGMA cluster diagrams (trees)
were generated using Jukes–Cantor distances in Geneious Prime using only sequences
newly generated in this study. The trees revealed clusters of specimens with low levels of
molecular variation, which were used as indicators of sequence-based species. The UPGMA
trees were used to estimate how many species were sampled in the NBA, as determined by
each genetic marker (rbcL-3P and UPA). To compare phylogenetic relationships between
our generated sequence data and published data in GenBank, RAxML trees [
40
] were
generated from MUSCLE alignments in Geneious Prime. Bootstrap values were calculated
by setting the algorithm iteration count to 1000. The RAxML trees were generated using
our unique sequences and GenBank sequences of the following categories: closest BLAST
search result [
41
], ceramialean species reported from the NBA, ceramialean species that are
found south of the NBA and may expand their ranges into the NBA as a result of climate
change or other vectors, and ceramialean species that are known to be introduced outside
their native ranges.
2.5. Species Determinations
Species determinations were reached by combining BLAST search results of molecular
data with morphological examination based on the reference compendia mentioned above.
Molecular results were determined simply by using the BLAST search engine to compare
newly generated sequences. The closest match by percent identity was generally used
to confirm genus assignments, and where the percent identity was greater than 99% or
99.5% for rbcL-3P and UPA, respectively, the same species was presumed (see Section 3.3
for assessment of molecular variation). In many instances, we generated the first sequences
for a certain species for either the UPA or rbcL-3P marker, so the closest BLAST search
results were not in fact the same species as the species we were searching. Historical reports
that included descriptions of the morphology and ecology of algae were used to reconcile
molecular species determinations.
3. Results
3.1. Sequencing
A total of 415 sequences were generated from 268 specimens of red algae of the order
Ceramiales in the Narragansett Bay Area (NBA), including 203 rbcL-3P sequences and
Diversity 2024,16, 554 6 of 42
212 UPA sequences. The UPA marker, in general, had much higher amplification and
sequence success than that of the rbcL-3P primers used (Table 4). In fact, over 40 more
UPA sequences were generated than rbcL-3P sequences, even though over 1.5 times as
many rbcL-3P PCR experiments were carried out than UPA PCR experiments. Generally,
PCR experiments using the UPA primers yielded amplicons of up to fivefold greater
concentrations than PCR experiments using the rbcL-3P primers, even when amplifying
the same DNA extract.
Table 4. Summary of PCR and sequencing success. Fractions show successes over attempts and the
percent success.
Primer Pair PCR Sequencing #Sequences
UPA-F/UPA-R 185/198 (93.4%) 136/172 (79.1%) 133
F753/R1442 204/294 (69.4%) 101/169 (59.8%) 91
F753/rbcLrevNEW 48/61 (78.7%) 17/31 (54.8%) 17
3.2. Molecular Biodiversity of Ceramiales
A total of 37 species were molecularly validated from the NBA, including 2 potentially
undescribed species and at least one new report of an introduced species in the NBA
(Figure 2). Of the 37 molecularly validated species, 32 were validated with the rbcL-3P
marker, 36 were validated with the UPA marker, and 31 were validated with both markers
(Figure 3A,B, Table 5). The availability of rbcL-3P sequence data in GenBank is robust; how-
ever, this study generated the first rbcL-3P sequences for four species (Table 5). Conversely,
there were far less published ceramialean UPA sequence data to which our sequences could
be compared, and this study generated the first UPA sequences for 21 species (Table 5).
Diversity 2024, 16, x FOR PEER REVIEW 7 of 43
sequences could be compared, and this study generated the first UPA sequences for 21
species (Table 5).
Figure 2. Venn diagram summarizing all species historically reported from the NBA and those that
we encountered. Unnumbered bullets indicate species reported from the NBA that are synonyms of
the above name (syn.), previously misidentified, but are now recognized as the above name (prev.),
or are included in the species group of the above name (inc.).
In most cases, morphological species identities based on historical reports were con-
sistent with molecular species identities (Figure 4). However, there were six taxa where
molecular species determinations were inconsistent with historical morphological reports
(Figure 4, gray triangles). Along with the taxonomic inconsistencies, another five species
were collected in this study, which have not been previously reported in the NBA at all
(Figures 2 and 4, black circles). The species treatment section below includes descriptions
and detailed discussion of these 11 taxa.
Figure 2. Venn diagram summarizing all species historically reported from the NBA and those that
we encountered. Unnumbered bullets indicate species reported from the NBA that are synonyms of
the above name (syn.), previously misidentified, but are now recognized as the above name (prev.),
or are included in the species group of the above name (inc.).
Diversity 2024,16, 554 7 of 42
Diversity 2024, 16, x FOR PEER REVIEW 8 of 43
Figure 3. UPGMA phylogenetic trees showing species of the order Ceramiales based on sequence
data generated in this study. (A) rbcL-3P. (B) UPA. A total of 32 species were validated with the
rbcL-3P marker, 36 were validated with the UPA marker, and 31 were validated with both. Species
names are colored by family: yellow—Callithamniaceae; red—Ceramiaceae; light blue—Dasyaceae;
dark blue—Delesseriaceae; pink—Rhodomelaceae; cyan—Spyridiaceae; green—Wrangeliaceae.
The n-values indicate how many sequences were generated for that particular species. Black stars
indicate species for which we have sequence data for only one DNA marker (e.g., no UPA sequences
were generated for Ceramium plenatunicum).
While molecular data was generated for 37 species, about 47 species were reported
from the NBA based on historical records and reconciliation of these reports with molec-
ular data (Figure 2). The number of historically reported names from the NBA is an ap-
proximate estimate, since there have been a variety of complicated taxonomic changes in
the ceramialean flora over the past several decades. To give a few examples of these com-
plications, many of these names were erroneously applied to taxa, in some cases calling a
single highly morphologically plastic species multiple different names [42], in some cases
not recognizing the cryptic diversity of a single species complex and compiling multiple
species under one name [43], or applying an extralimital name to a morphologically sim-
ilar taxon [44–46]. Of the 47 species reported from the NBA, about 15 species were not
collected during the summer of 2023 or molecularly validated through aempts to DNA
barcode RWU herbarium presses (Figure 2). Most of the 15 species that were unaccounted
for were either winter annuals or deeper-water algae that would not have been encoun-
tered during the summertime shallow intertidal snorkeling collections.
Figure 3. UPGMA phylogenetic trees showing species of the order Ceramiales based on sequence
data generated in this study. (A) rbcL-3P. (B) UPA. A total of 32 species were validated with the
rbcL-3P marker, 36 were validated with the UPA marker, and 31 were validated with both. Species
names are colored by family: yellow—Callithamniaceae; red—Ceramiaceae; light blue—Dasyaceae;
dark blue—Delesseriaceae; pink—Rhodomelaceae; cyan—Spyridiaceae; green—Wrangeliaceae. The
n-values indicate how many sequences were generated for that particular species. Black stars indicate
species for which we have sequence data for only one DNA marker (e.g., no UPA sequences were
generated for Ceramium plenatunicum).
In most cases, morphological species identities based on historical reports were con-
sistent with molecular species identities (Figure 4). However, there were six taxa where
molecular species determinations were inconsistent with historical morphological reports
(Figure 4, gray triangles). Along with the taxonomic inconsistencies, another five species
were collected in this study, which have not been previously reported in the NBA at all
(Figures 2and 4, black circles). The species treatment section below includes descriptions
and detailed discussion of these 11 taxa.
Diversity 2024,16, 554 8 of 42
Table 5. Overview of sequence data generated in this study, including the number of rbcL-3P and UPA
sequences generated, overlap (the number of specimens for which both rbcL-3P and UPA sequences
were generated), and accession numbers for published data generated in this study. Asterisks (*)
indicate species for which this study generated the first sequences for a particular marker. Gray-
highlighted rows indicate species discussed in greater detail in the species treatments. Hyperlinks to
iNaturalist observations of representative specimens with images of sequenced algae are embedded
in the species names. All linked images show specimens that were sequenced in this study, with
the exception of the linked observation of Spermothamnion repens, showing a specimen that was not
sequenced but is representative of the species.
Family Species # of rbcL-3P # of UPA Overlap rbcL-3P Acc. #
rbcL-3P MARI #
UPA Acc. # UPA MARI #
Callithamniaceae—4 spp. 13 22 13
Aglaothamnion halliae 7 7 * 7 PP805876 MARI-04397 PP862934 MARI-04399
Callithamnion corymbosum 6 9 6 PP805878 MARI-04464 PP862957 MARI-04642
Callithamnion tetragonum 0 5 * 0 --- --- PP862946 MARI-04554
Seirospora interrupta 0 1 * 0 --- --- PP862927 MARI-03581
Ceramiaceae—9 spp. 44 39 29
Antithamnion hubbsii 5 6 4 PP805883 MARI-04509 PP862942 MARI-04511
Antithamnion sp. 2 * 3 * 2 PP805891 MARI-04595 PP862950 MARI-04595
Antithamnionella floccosa 0 1 * 0 --- --- PP862925 MARI-02643
Antithamnionella spirographidis
1 * 1 * 1 PP805894 MARI-04622 PP862952 MARI-04622
Ceramium facetum 12 9 8 PP805885 MARI-04534 PP862936 MARI-04418
Ceramium plenatunicum 3 0 0 PP805884 MARI-04529 --- ---
Ceramium secundatum 14 11 * 9 PP805877 MARI-04440 PP862954,
PP862958
MARI-04626,
MARI-04513
Ceramium virgatum 3 2 * 2 PP805871 MARI-04173 PP862931 MARI-04173
Ceramothamnion translucidum 4 5 3 PP805882 MARI-04471 PP862940 MARI-04471
Dasyaceae—3 spp. 8 14 8
Dasya cf. elegans 2 2 2 PP805874 MARI-04392 PP862935 MARI-04407
Dasya pedicellata 3 6 3 PP805893 MARI-04604 PP862955,
PP862959
MARI-04628,
MARI-04604
Dasysiphonia japonica 3 4 3 PP805875 MARI-04393 PP862933 MARI-04393
Delesseriaceae—2 spp. 1 5 1
Grinnellia americana 1 3 * 1 PP805892 MARI-04597 PP862951 MARI-04597
Phycodrys sp. 0 2 * 0 --- --- PP862926 MARI-02955
Rhodomelaceae – 14 spp. 123 115 81
Acanthosiphonia echinata 1 1 * 1 PP805886 MARI-04540 PP862943 MARI-04540
Bostrychia radicans 2 5 * 2 PP805870 MARI-04163 PP862930 MARI-04169
Carradoriella elongata 3 3 2 PP805872 MARI-04185 PP862945 MARI-04547
Chondria atropurpurea 7 9 * 5 PP805879 MARI-04465 PP862937 MARI-04465
Chondria baileyana 14 15 * 10 PP805887 MARI-04545 PP862944 MARI-04545
Chondria littoralis/sedifolia 10 12 * 10 PP805888 MARI-04557 PP862947 MARI-04556
Kapraunia schneideri 6 7 5 PP805865 MARI-01246 PP862929 MARI-04168
Melanothamnus spp. 44 39 29 --- --- --- ---
Polysiphonia stricta 7 4 1 PP805869 MARI-02324 PP862928 MARI-04157
Rhodomela sp. 0 1 0 --- --- PP862924 MARI-02262
Streblocladieae sp. 1 * 1 * 1 PP805880 MARI-04466 PP862938 MARI-04466
Vertebrata fucoides 18 9 * 7 PP805867,
PP805873
MARI-01882,
MARI04391 PP862941 MARI-04482
Vertebrata lanosa 6 6 5 PP805868 MARI-02045 PP862932 MARI-04181
Vertebrata nigra 4 3 * 3 PP805895 MARI-04624 PP862953 MARI-04624
Spyridiaceae—1 sp. 6 3 3
Spyridia americana 6 3 * 3 PP805881 MARI-04469 PP862939 MARI-04469
Wrangeliaceae—4 spp. 8 14 7
Griffithsia globulifera 4 * 8 * 4 PP805889 MARI-4581 PP862948 MARI-04582
Pleonosporium novae-angliae 1 3 * 1 PP805890 MARI-04590 PP862949 MARI-04591
Plumaria plumosa 1 2 1 PP805896 MARI-04636 PP862956 MARI-04636
Spermothamnion repens 2 1 1 PP805866 MARI-01818 PP862923 MARI-02047
Diversity 2024,16, 554 9 of 42
Diversity 2024, 16, x FOR PEER REVIEW 9 of 43
Figure 4. RAxML phylogenetic trees showing all species barcoded in this study, closest BLAST
search results in GenBank, species historically reported from the NBA, and any other contextually
relevant species. (A) rbcL-3P. (B) UPA. Sequences generated in this study start with the five-digit
MARI number and end with their GenBank accession numbers; outsourced sequences do not start
with a MARI number. Gray triangles indicate species with discrepancies between morphological
and molecular species identities, and black circles indicate species that are newly reported in this
study. Note that there are 6 gray triangles in the rbcL-3P tree (A) and only five in the UPA tree (B)
because no UPA sequences were generated for Ceramium plenatunicum. Species marked by colored
shapes are discussed in the species treatments.
Figure 4. RAxML phylogenetic trees showing all species barcoded in this study, closest BLAST
search results in GenBank, species historically reported from the NBA, and any other contextually
relevant species. (A)rbcL-3P. (B) UPA. Sequences generated in this study start with the five-digit
MARI number and end with their GenBank accession numbers; outsourced sequences do not start
with a MARI number. Gray triangles indicate species with discrepancies between morphological and
molecular species identities, and black circles indicate species that are newly reported in this study.
Note that there are 6 gray triangles in the rbcL-3P tree (A) and only five in the UPA tree (B) because
no UPA sequences were generated for Ceramium plenatunicum. Species marked by colored shapes are
discussed in the species treatments.
Diversity 2024,16, 554 10 of 42
While molecular data was generated for 37 species, about 47 species were reported
from the NBA based on historical records and reconciliation of these reports with molecular
data (Figure 2). The number of historically reported names from the NBA is an approxi-
mate estimate, since there have been a variety of complicated taxonomic changes in the
ceramialean flora over the past several decades. To give a few examples of these compli-
cations, many of these names were erroneously applied to taxa, in some cases calling a
single highly morphologically plastic species multiple different names [
42
], in some cases
not recognizing the cryptic diversity of a single species complex and compiling multiple
species under one name [
43
], or applying an extralimital name to a morphologically similar
taxon [
44
–
46
]. Of the 47 species reported from the NBA, about 15 species were not collected
during the summer of 2023 or molecularly validated through attempts to DNA barcode
RWU herbarium presses (Figure 2). Most of the 15 species that were unaccounted for were
either winter annuals or deeper-water algae that would not have been encountered during
the summertime shallow intertidal snorkeling collections.
3.3. Molecular-Assisted Identification of Ceramialean Species in the NBA
The barcoding gap for separating species using the rbcL-3P marker is around 1.00%
(7 base pairs), which separated the close pair of Dasya elegans and Dasya pedicellata. However,
all other similar species pairs in this study were over 2% (14 base pairs) divergent from
one another for the rbcL-3P marker. The barcoding gap for the UPA marker is about 0.50%
(2 base pairs), which was the interspecies divergence between Ceramium secundatum and
Ceramium virgatum, but again all other species pairs were over 1% (4 base pairs) divergent
from one another. It is to be noted, though, that in most cases, if the interspecies divergence
for one marker was particularly low, this was not the case for the other marker. For
example, the minimum interspecies divergence between D. elegans and D. pedicellata was
1.00% (7 base pairs) for rbcL-3P, but it was 1.63% (6 base pairs) for UPA. Similarly, where
the minimum interspecies divergence was 0.54% (2 base pairs) between C. secundatum and
C. virgatum for the UPA marker, it was no less than 4.24% (28 base pairs) for the rbcL-3P
marker. Using both markers together provided a helpful check to determine if two genetic
groups were the same species or not, which was useful for informing species identifications.
Molecular species identifications between the two markers (UPA and rbcL-3P) were
100% in accordance with one another. This appeared to be the case even when the rbcL-3P
and UPA sequences for a given specimen did not always have the same closest BLAST
search results (Table 6). This seemed to be a result of one of two things depending on the
species: no sequence data for that species were available for one or both markers, so the clos-
est BLAST search results were inherently inconsistent between the markers (e.g., the rbcL-3P
sequence for MARI-04622 matched Antithamnionella spirographidis, but the UPA sequence
was closest to Antithamnionella ternifolia since there is no Antithamnionella spirographidis UPA
sequence data available), or there is some taxonomic ambiguity or potentially misnamed
sequences in GenBank (this appeared to be the case only for Dasya spp. and Melanothamnus
spp.). The rbcL-3P marker was found to be similarly conserved between individuals of a
species with respect to the UPA marker, as the intraspecies divergence of rbcL-3P was at
most 0.30% (2 base pair divergence; this occurred in Callithamnion corymbosum and Vertebrata
fucoides; Table 6), and the UPA marker had a maximum intraspecies divergence of 0.31%
(1 base pair divergence; this occurred in multiple taxa; Table 6). However, as mentioned
above, the lowest interspecies divergences for both markers were above these thresholds,
with the lowest rbcL-3P interspecies divergence being between Dasya elegans and Dasya
pedicellata at 1.00% (7 base pairs), and for UPA it was 0.54% (2 base pairs) between Ceramium
secundatum and Ceramium virgatum. The Melanothamnus species group was excluded from
these analyses, since this group includes a few hybridized species in the Northwest Atlantic
and displays high genetic variance [43].
Diversity 2024,16, 554 11 of 42
Table 6. Overview of molecular species determinations of all species validated in this study, including
nearest GenBank BLAST search results (percent identity to closest match, species, and GenBank
accession number) and intraspecies divergences for each marker. Gray-highlighted rows indicate
species discussed in greater detail in the species treatments. BLAST search results are not included
for Melanothamnus spp., since this group has a large sequence divergence and matches numerous
different species within the genus.
Family Species rbcL-3P
(% ID) Nearest BLAST UPA (% ID) Nearest BLAST rbcL-3P Intra.
Div. UPA Intra. Div
Callithamniaceae—
4 spp.
Aglaothamnion
halliae 100%
Aglaothamnion
halliae
AF439305
98.10% Aglaothamnion sp.
KY573954 0% 0%
Callithamnion
corymbosum 99.40%
Callithamnion
corymbosum
DQ110896
100%
Callithamnion
corymbosum
KC795892
0–0.30% 0–0.28%
Callithamnion
tetragonum --- --- 97.83%
Callithamnion
tetragonum
MK814616
--- 0%
Seirospora
interrupta --- --- 94.86%
Cryptopleura
ramosa
MK814633
--- ---
Ceramiaceae—9
spp.
Antithamnion
hubbsii 100%
Antithamnion
hubbsii
KJ202093
100%
Antithamnion
hubbsii
KJ202103
0% 0%
Antithamnion sp. 96.40%
Antithamnion
kylinii
JN089393
98.37%
Antithamnion
hubbsii
KJ202103
0% 0%
Antithamnionella
floccosa --- --- 99.19%
Antithamnionella
ternifolia
MK814608
--- ---
Antithamnionella
spirographidis 100%
Antithamnionella
spirographidis
DQ022810
99.19%
Antithamnionella
ternifolia
MK814608
--- ---
Ceramium facetum 99.85%
Ceramium
diaphanum
KF367765
100%
Ceramium
diaphanum
KF367765
0–0.07% 0–0.28%
Ceramium
plenatunicum 98.80% Ceramium derbesii
FR775779 --- --- 0% ---
Ceramium
secundatum 100%
Ceramium
secundatum
DQ110904
98.92%
Ceramium
diaphanum
KF367775
0% 0%
Ceramium
virgatum 100%
Ceramium
virgatum
KT250272
98.38%
Ceramium
diaphanum
KF367775
0.07–0.22% 0%
Ceramothamnion
translucidum 100% “Ceramium sp. 2”
KF367768 100% “Ceramium sp. 2”
KF367781 0–0.15% 0%
Dasyaceae—3
spp.
Dasya cf. elegans 100%
“Dasya sp. 1
baillouviana”
MW698713
98.64% Dasya sp.
HQ421299 0% 0%
Dasya pedicellata 100% Dasya pedicellata
ON002436 100% Dasya baillouviana
HQ421392 0% 0%
Dasysiphonia
japonica 100%
Dasysiphonia
japonica
MH287465
100%
Dasysiphonia
japonica
MK814640
0% 0%
Delesseriaceae—
2 spp.
Diversity 2024,16, 554 12 of 42
Table 6. Cont.
Family Species rbcL-3P
(% ID) Nearest BLAST UPA (% ID) Nearest BLAST rbcL-3P Intra.
Div. UPA Intra. Div
Grinnellia
americana 100%
Grinnellia
americana
AF254184
97.83%
Membranoptera
tenuis
NC_032399
--- 0%
Phycodrys sp. --- --- 100% Phycodrys radicosa
KC795887 --- 0%
Rhodomelaceae—
14 spp.
Acanthosiphonia
echinata 100%
Acanthosiphonia
echinate
MF120866
97.57%
Polysiphonia
binneyi
KY573931
--- ---
Bostrychia radicans
100%
Bostrychia radicans
AY920882 96.48%
Bostrychia
moritziana
NC_035266
0.30% 0%
Carradoriella
elongata 100%
Carradoriella
elongata
MF120875
99.73%
Carradoriella
elongata
NC_035274
0% 0%
Chondria
atropurpurea 100%
Chondria
atropurpurea
MH388516
98.10% Chondria sp.
MF101431 0–0.08% 0%
Chondria baileyana 100% Chondria baileyana
KU564500 97.83% Chondria sp.
OM468962 0–0.22% 0–0.28%
Chondria
littoralis/sedifolia 100% Chondria littoralis
KF672853 97.02% Chondria sp.
MF101429 0–0.15% 0–0.27%
Kapraunia
schneideri 100%
Kapraunia
schneideri
MT597079
99.19%
Kapraunia
schneideri
NC_035296
0–0.29% 0%
Melanothamnus
spp. --- --- --- --- 0–1.94% 0–0.73%
Polysiphonia stricta
99.85%
Polysiphonia stricta
EU492916 100%
Polysiphonia stricta
MF101428 0% 0–0.31%
Rhodomela sp. --- --- 100%
Rhodomela
confervoides
NC_035271
--- ---
Streblocladieae
sp. 93.30%
Kapraunia
pentamera
HM573564
97.83% Polysiphonia sp.
HQ421052 --- ---
Vertebrata fucoides 100% Vertebrata fucoides
EU492913 98.37% Vertebrata isogona
NC_035278 0–0.30% 0%
Vertebrata lanosa 100% Vertebrata lanosa
KU564487 100% Vertebrata lanosa
KP208097 0% 0%
Vertebrata nigra 100% Vertebrata nigra
MF120893 98.37% Vertebrata isogona
NC_035278 0% 0%
Spyridiaceae—1
sp.
Spyridia americana 100% Spyridia americana
MW770750 99.19%
Spyridia
filamentosa
HQ421086
0–0.15% 0%
Wrangeliaceae—
4 spp.
Griffithsia
globulifera 89.66% Griffithsia okiensis
EU195056 95.93% Plumaria plumosa
MK814703 0–0.16% 0%
Pleonosporium
novae-angliae 96.20%
Lophothamnion
comatum
KU381977
99.18%
Aglaothamnion
boergesenii
HQ421367
--- 0%
Plumaria plumosa 100% Plumaria plumosa
KU381993 99.19% Plumaria plumosa
MK814703 --- 0.27%
Spermothamnion
repens 100%
Spermothamnion
repens
MK814735
100%
Spermothamnion
repens
MK814735
0.15% ---
Diversity 2024,16, 554 13 of 42
For species that had very close neighbors, such as species of the genera Ceramium,
Dasya, or Vertebrata, intraspecies divergences for both markers were well below those
genetic distances (percent identity) to the nearest neighbor. For example, a close pair such
as C. secundatum and C. virgatum, which were at minimum 0.54% (2 base pairs) divergent,
had a 0% intraspecies divergence (Table 6).
Since the UPA marker had an established barcoding gap that was reliable and had
species clusters and identifications consistent with the rbcL-3P marker, the UPA marker
was found to be just as effective as the rbcL-3P marker in identifying species of the order
Ceramiales. This is consistent with other studies that have used the UPA marker for
molecular surveys of red algae [47,48].
There were two taxa for which sequence data were challenging to obtain. These species
that posed challenges during the barcoding process included Callithamnion tetragonum and
Spermothamnion repens. The former posed no challenges when working with the UPA
primers; in fact, complete sequences were generated for five C. tetragonum specimens.
However, no rbcL-3P sequences were generated for C. tetragonum. Considering the success
with the UPA marker, the lack of success with the rbcL-3P primers was likely a result of
the primers not being a good base pair match to their complements in the C. tetragonum
genome. Spermothamnion repens, on the other hand, seemed to be much more challenging
to obtain usable DNA extracts using the BioLine extraction method. The only sequence
data generated for this species were from older DNA extracts that had been prepared by
grinding liquid nitrogen frozen material with a mortar and pestle.
3.4. Species Treatments
Most taxa sampled in this study have been reported from the NBA historically, and
both the morphological and molecular species determinations agree with the reports of
these species’ identities and presence in the NBA (Figure 2). However, 11 species of
interest are discussed below, namely new reports for the area and species for which there
are discrepancies between the molecular results, morphological results, and historical
documentation.
3.4.1. New Reports for the Narragansett Bay Area (5 Species)
Acanthosiphonia echinata (Harvey) Savoie & G. W. Saunders
Molecular Results: Both rbcL-3P and UPA sequences were generated for a single
specimen, MARI-04540 (Table 5). The rbcL-3P and UPA sequences were a 100% match to
Acanthosiphonia echinata MF120866 (Figure 5A) from New Brunswick and a 97.57% match to
Polysiphonia binneyi KY573931 from Pacific Panama (Figure 5B), respectively. Both markers
supported Carradoriella elongata as being the nearest genetic neighbor that we collected,
and MARI-04540 differed from C. elongata by 7.81–8.04% and 3.51–3.68% for rbcL-3P and
UPA, respectively.
Locality and Morphology: One specimen, MARI-04540, was collected from drift
material on 29 July 2023 along the open coast at Horseneck Beach, Westport, MA, USA
(Table 7). The thalli were small, matting epiphytes no more than 30 mm tall that covered
a strand of the Chorda filum (Figure 6A,B). Branching was alternate to irregular, with
adventitious branching being common and giving the thallus an echinate, “spine-like”
appearance (Figure 6C,D). Filaments were erect, lacking any prostrate systems, and arose
from discoid, rhizoidal holdfasts (Figure 6E). Axes were ecorticate throughout, and branches
were borne from cell lineage nodes (Figure 6F,G). The axes were typically shaped such
that cells would be slightly concave and the axes would be wider at internodes between
segments in older axes (Figure 6H), but sometimes, cells were inflated near branchlet tips,
giving the appearance of corn on the cob (Figure 6I,J). The axes had four pericentral cells
throughout, as seen in the cross-section (Figure 6J,K). Trichoblasts were abundant, colorless,
highly branched, and present at the branchlet tips (Figure 6M). At least some of the material
collected was cystocarpic, but there may have been multiple reproductive stages present in
the mix (Figure 6N). The morphology described above, particularly the echinate branching
Diversity 2024,16, 554 14 of 42
pattern, ecorticate character, and four pericentral cells, is consistent with the description of
A. echinata [49].
Diversity 2024, 16, x FOR PEER REVIEW 15 of 43
Mexico within the last two decades. There are a few possibilities for this trend. A. echinata
may have recently been introduced outside its range up the east coast of North America
and elsewhere in the world, which would explain why it has only recently been reported
in so many other locations outside of the region of the type locality. The introduction of
the species to the Mediterranean and Indonesia would likely have been enabled by an-
thropogenic means, but the spread of A. echinata up the east coast of North America could
be a natural consequence of the species extending its range as a result of global warming,
making northerly waters more habitable to subtropical species in the summer [13]. There
is also another possible reason that this species has only recently been recognized in new
habitats, and that could be because it is, at first glance, very similar to a variety of other
ceramialean species (e.g., Melanothamnus spp. or Neosiphonia spp.). The increased popu-
larity of molecular surveying methods, such as those employed here, may have been the
reason A. echinata was identified in these new regions only within the last two decades
and not earlier, since this species is morphologically similar to many other species. Further
examination of older herbarium material may elucidate whether A. echinata has been pre-
sent in places such as the NBA for a while and has been overlooked due to its easily mis-
takable identity, or if it truly was not present in these waters until recently.
Figure 5. RAxML phylogenetic trees of Rhodomelaceae and Dasyaceae, focusing on species discussed
in the species treatments. (A)rbcL-3P. (B) UPA. Closely related taxa and other published sequences
were included as context. Sequences generated in this study start with the 5-digit MARI number and
end with their GenBank accession numbers. Outsourced sequences do not start with a MARI number.
Remarks: Based on the molecular results and morphology, this species was identified
as A. echinata. This is the first report of A. echinata from the NBA and the surrounding
regions (Figure 2). The type locality of the species is Florida, and it has been reported
widely throughout the Caribbean and Gulf of Mexico, but it has recently been introduced
to the Mediterranean and Indonesia [
16
,
50
,
51
]. It has also recently been reported in North
Carolina and New Brunswick, but not anywhere in between [
49
,
52
]. However, just as with
Ceramothamnion translucidum, considering that A. echinata has been reported in Florida,
North Carolina, and New Brunswick, it would follow that A. echinata would also be present
in the NBA since the NBA is within the established range of the species. However, A.
echinata has only been reported outside its original range in the Caribbean and Gulf of
Mexico within the last two decades. There are a few possibilities for this trend. A. echinata
Diversity 2024,16, 554 15 of 42
may have recently been introduced outside its range up the east coast of North America
and elsewhere in the world, which would explain why it has only recently been reported
in so many other locations outside of the region of the type locality. The introduction
of the species to the Mediterranean and Indonesia would likely have been enabled by
anthropogenic means, but the spread of A. echinata up the east coast of North America could
be a natural consequence of the species extending its range as a result of global warming,
making northerly waters more habitable to subtropical species in the summer [
13
]. There
is also another possible reason that this species has only recently been recognized in new
habitats, and that could be because it is, at first glance, very similar to a variety of other
ceramialean species (e.g., Melanothamnus spp. or Neosiphonia spp.). The increased popularity
of molecular surveying methods, such as those employed here, may have been the reason A.
echinata was identified in these new regions only within the last two decades and not earlier,
since this species is morphologically similar to many other species. Further examination
of older herbarium material may elucidate whether A. echinata has been present in places
such as the NBA for a while and has been overlooked due to its easily mistakable identity,
or if it truly was not present in these waters until recently.
Diversity 2024, 16, x FOR PEER REVIEW 16 of 43
Figure 5. RAxML phylogenetic trees of Rhodomelaceae and Dasyaceae, focusing on species dis-
cussed in the species treatments. (A) rbcL-3P. (B) UPA. Closely related taxa and other published
sequences were included as context. Sequences generated in this study start with the 5-digit MARI
number and end with their GenBank accession numbers. Outsourced sequences do not start with a
MARI number.
Figure 6. Acanthosiphonia echinata epiphytic on Chorda filum collected in the drift at Horseneck Beach,
Westport, MA, USA. (A) The light golden epiphytes covered a strand of Chorda filum. (B–D) Detail
of echinate branching paerns. (E) Basal rhizoidal disk of an erect filament. (F–H) Axes are ecorticate
throughout, and segments are usually 1–1.5 diameters long. (I) Detail of the distal portion of a
branchlet with “spine-like” adventitious branchlets (arrowheads). (J) Some distal axes had very
swollen cells that give the appearance of corn on the cob. (K,L) Axes have 4 pericentral cells (num-
bered) throughout. (M) Branchlet terminus with sparse but present trichoblasts. (N) A young cys-
tocarp developing on a short stalk.
Tab l e 7. Ecological data of molecularly validated Ceramiales from the NBA. Most of these data are
from specimens collected in the summer and fall of 2023, with some data of older specimens from
numerous other sites that were also sequenced in this study. Asterisks (*) indicate data that are not
molecularly validated but are strong morphological matches to sequenced specimens. (Key: BH:
Bristol Harbor, Bristol, RI; BM: Belmont Beach, Newport RI; BP: Black Point, Narraganse, RI; BSP:
Brenton State Park, Newport, RI; BTP: Beavertail Point, Jamestown, RI; CRN: Cormorant Rock,
Newport, RI; CSP: Colt State Park, Bristol, RI; CWB: Cherry & Webb Beach, Westport, MA; EAS:
Easton’s Beach, Newport, RI; FBN: Fogland Beach North, Tiverton, RI; FBS: Fogland Beach South,
Tiverton, RI; FW: Fort Wetherill, Jamestown, RI; GB: Garbage Beach, Falmouth, MA; GOO: Goose-
berry Beach, Newport, RI; HHP: High Hill Point, Tiverton, RI; HNB: Horseneck Beach, Westport,
MA; KB: King’s Beach, Newport, RI; KR: Kickemuit River, Bristol, RI; LHB: Lighthouse Beach,
Figure 6. Acanthosiphonia echinata epiphytic on Chorda filum collected in the drift at Horseneck
Beach, Westport, MA, USA. (A) The light golden epiphytes covered a strand of Chorda filum.
(B–D) Detail of echinate branching patterns. (E) Basal rhizoidal disk of an erect filament. (F–H) Axes
are ecorticate throughout, and segments are usually 1–1.5 diameters long. (I) Detail of the distal
portion of a branchlet with “spine-like” adventitious branchlets (arrowheads). (J) Some distal axes
had very swollen cells that give the appearance of corn on the cob. (K,L) Axes have 4 pericentral cells
(numbered) throughout. (M) Branchlet terminus with sparse but present trichoblasts. (N) A young
cystocarp developing on a short stalk.
Diversity 2024,16, 554 16 of 42
Table 7. Ecological data of molecularly validated Ceramiales from the NBA. Most of these data
are from specimens collected in the summer and fall of 2023, with some data of older specimens
from numerous other sites that were also sequenced in this study. Asterisks (*) indicate data that
are not molecularly validated but are strong morphological matches to sequenced specimens. (Key:
BH: Bristol Harbor, Bristol, RI; BM: Belmont Beach, Newport RI; BP: Black Point, Narragansett, RI;
BSP: Brenton State Park, Newport, RI; BTP: Beavertail Point, Jamestown, RI; CRN: Cormorant Rock,
Newport, RI; CSP: Colt State Park, Bristol, RI; CWB: Cherry & Webb Beach, Westport, MA; EAS:
Easton’s Beach, Newport, RI; FBN: Fogland Beach North, Tiverton, RI; FBS: Fogland Beach South,
Tiverton, RI; FW: Fort Wetherill, Jamestown, RI; GB: Garbage Beach, Falmouth, MA; GOO: Gooseberry
Beach, Newport, RI; HHP: High Hill Point, Tiverton, RI; HNB: Horseneck Beach, Westport, MA; KB:
King’s Beach, Newport, RI; KR: Kickemuit River, Bristol, RI; LHB: Lighthouse Beach, Chatham, MA;
MAC: Mackerel Cove, Jamestown, RI; NMS: Northeastern Marine Science Center, Nahant, MA; NP:
Ninigret Pond, Charlestown, RI; NTB: Narragansett Town Beach, Narragansett, RI; PB: Pebble Beach,
Rockport, MA; PLC: Police Cove, Barrington, RI; RWU: Roger Williams University Waterfront, Bristol,
RI; SCB: Second Beach, Middletown, RI; SP: Sakonnet Point, Little Compton, RI; SPG: Sandy Point,
Greenwich, RI).
Families and Species Dates Collected Substratum Habitat Localities
Callithamniaceae—4 spp.
Aglaothamnion halliae JUN Epiphytic on Gracilaria,
Spartina, and shells
Low intertidal to shallow
subtidal, estuarine,
common in June
RWU
Callithamnion corymbosum MAY–OCT, NOV *, DEC *
Most often lithophytic,
occasionally epiphytic on
coarse algae
Low intertidal to subtidal,
estuarine, common
BH, CSP *, HHP, NP, PLC
*, SPG
Callithamnion tetragonum MAR, JUN–OCT
Epiphytic on Codium,
Chondrus and other coarse
algae
Lower intertidal, subtidal,
tidepools, open coast,
more abundant JUL—OCT
BSP, BTP, FW, KB, SCB *
Seirospora interrupta AUG (Drift) Estuarine GB
Ceramiaceae—9 spp.
Antithamnion hubbsii JUN–OCT
Epiphytic on Phyllophora,
Chondrus, and other coarse
algae
Subtidal, open coast with
high wave action,
common annuals with
new growth appearing in
June and larger growth in
fall
BP, CRN, FW, KB, SCB *
Antithamnion sp. AUG–SEP Epiphytic on coarse algae
and shells
Subtidal, open coast to
unprotected estuarine
waters, uncommon
FBS, KB
Antithamnionella floccosa APR Epiphytic on jetty
“Common, exposed area 2
feet above datum” PB
Antithamnionella
spirographidis OCT (Drift) Open coast LHB
Ceramium facetum MAY–SEP
Mostly lithophytic,
occasionally epiphytic on
Gracilaria
Upper intertidal, shallow
subtidal, estuarine and
open coast, common
BH, BP, FBN, FW, PLC,
RWU
Ceramium plenatunicum AUG Epiphytic on Gracilaria
Low intertidal to shallow
subtidal, protected
estuarine, abundant at this
site
FBN
Ceramium secundatum MAY–NOV Epiphytic on coarse algae
or lithophytic
Subtidal, mostly open
coast but occasionally in
estuarine drift, common
BP, BSP, FW, HNB, KB,
LHB, RWU
Ceramium virgatum MAY–AUG Epiphytic on coarse algae
or lithophytic
Subtidal, open coast,
uncommon FW, SCB
Ceramothamnion
translucidum JUN–SEP
Epiphytic on Fucus and
other coarse and mixed
with soft algae
On algae growing on
floating docks or attached
to drift algae, estuarine
and open coast,
inconspicuous but
common
HNB, NP, SP
Dasyaceae—3 spp.
Diversity 2024,16, 554 17 of 42
Table 7. Cont.
Families and Species Dates Collected Substratum Habitat Localities
Dasya cf. elegans MAY–JUN Lithophytic Low intertidal to subtidal,
estuarine RWU
Dasya pedicellata JUL, SEP–NOV
Mostly lithophytic, but
younger thalli epiphytic
on coarse algae and
Zostera
Subtidal, open coast,
common BM, HNB, KB, LHB
Dasysiphonia japonica JUN, JUL, AUG *–MAY * Lithophytic or epiphytic
on coarse algae
Low intertidal to subtidal,
estuarine and open coast,
common and widespread
BH *, BM *, CSP *, FW *,
HNB *, KB, NP, RWU, SCB
*
Delesseriaceae—2 spp.
Grinnellia americana JUN, JUL *, AUG *, OCT,
NOV *
Lithophytic, occasionally
on shells, once collected
on raphyrus mass of
Cliona celata
Subtidal, estuarine and
open coast, common and
widespread
CSP *, FBS, FW, PLC *,
RWU
Phycodrys sp. OCT, NOV (Drift) Subtidal, open coast BSP, NMS
Rhodomelaceae—14 spp.
Acanthosiphonia echinata JUL Epiphytic on Chorda in
drift
Probably subtidal, open
coast HNB
Bostrychia radicans JUN, JUL, NOV *
Mostly epiphytic on live
Geukensia, occasionally on
Spartina or spreading over
rocks
High intertidal, estuarine,
inconspicuous but
common at these sites
PLC, RWU
Carradoriella elongata
APR *, JUL, AUG *–OCT *,
NOV (Drift)
Probably subtidal, open
coast, new growth in April
and older growth growth
JUL–OCT, common
BM *, CWB *, HNB, KB *,
SCB
Chondria atropurpurea JUN–OCT Strictly lithophytic
Shallow subtidal, open
coast and estuarine,
widespread and common
BM *, FBN, FBS, HNB, KB,
NP, RWU, SCB
Chondria baileyana JUN–OCT
Lithophytic or epiphytic
on coarse algae and
Zostera
Low intertidal to shallow
subtidal and in tidepools,
estuarine and open coast,
widespread and common,
new growth appearing in
June, older growth in the
fall
BH, BP, EAS, FW, HNB,
KB, PLC, RWU
Chondria littoralis/sedifolia JUN–OCT
Mostly lithophytic, but
younger thalli epiphytic
on coarse algae and
Zostera
Subtidal, open coast, new
growth appearing in June,
older growth in the fall,
common
BM *, FW, HNB, KB, SCB
Kapraunia schneideri JUN–AUG Lithophytic or on pilings
or floating docks
Subtidal, estuarine or
open coast, widespread
and common
BH, BP *, PLC, RWU
Melanothamnus spp. APR–OCT, DEC *–MAR *
Lithophytic or epiphytic
on coarse algae or floating
docks
Mid intertidal to shallow
subtidal or in tidepools,
estuarine and open coast,
widespread and common
BH, BSP, BTP, CRN, CSP,
EAS, GOO, FW *, KB,
RWU, SCB, SPG
Polysiphonia stricta DEC *, FEB *, MAR–JUN Lithophytic
Mid intertidal to shallow
subtidal, new growth
appearing in December,
older growth present in
June.
BSP, CSP, FW, KB, RWU
Rhodomela sp. APR Lithophyte In tidepool, open coast BSP
Streblocladieae sp. JUN Lithophytic Shallow subtidal,
estuarine NP
Vertebrata fucoides FEB *, MAY, JUN, AUG *,
OCT Lithophytic
Subtidal, estuarine and
open coast, widespread,
common, very old growth
present in August
BSP, COR, CSP, GOO, FW,
KB, RWU, SCB, SPG
Vertebrata lanosa JUN, JUL *, AUG *,
SEP–NOV
Growing on Ascophyllum
nodosum
Mid intertidal to low
intertidal, open coast,
common
BP, GOO, FW, KB, SCB
Vertebrata nigra APR *, JUN, OCT Lithophytic Subtidal, open coast,
uncommon CWB *, GOO, FW *, LHB
Diversity 2024,16, 554 18 of 42
Table 7. Cont.
Families and Species Dates Collected Substratum Habitat Localities
Spyridiaceae—1 sp.
Spyridia americana JUN, JUL, AUG *–OCT *
Mostly lithophytic,
occasionally in tangled
masses with other algae
Subtidal, open coast and
estuarine, widespread,
common in certain
localities, older growth
present in October
KB *, NP, RWU
Wrangeliaceae—4 spp.
Griffithsia globulifera JUN, JUL, SEP
Mostly lithophytic,
occasionally epiphytic on
shells
Shallow subtidal,
estuarine, common in
certain localities
FBS, KR, PLC, RWU *
Pleonosporium novae-angliae
AUG, SEP Epiphytic on coarse algae
Subtidal, open coast,
uncommon, new growth
appearing in August
KB
Plumaria plumosa OCT (Drift) Open coast NTB
Spermothamnion repens JUN *–SEP *
Lithophytic or spreading
onto basal axes of coarse
algae
Subtidal, open coast,
common BP *, FW *, KB *, SCB *
Antithamnion sp.
Molecular Results: For two specimens, MARI-04535 and MARI-04595, both rbcL-3P
and UPA sequences were obtained, and another UPA sequence was generated from MARI-
04594. The rbcL-3P sequences were 0% divergent from each other, and their BLAST search
results were a 96.40% match to a cultured specimen of uncertain origin, Antithamnion kylinii
JN089393 (Figure 7A). The UPA sequences were also identical to one another, and their
BLAST search results were a 98.37% match to Antithamnion hubbsii from North Carolina
(Figure 7A). Both of these BLAST search results were more than 1% divergent from this
species, indicating that MARI-04535 and MARI-04595 are species not currently represented
molecularly in GenBank. The nearest molecular neighbor that we collected according to
both markers was Antithamnion hubbsii, which differed from this species by 6.15–6.30%
and 1.63% for rbcL-3P and UPA, respectively, meaning that this species is distinct from A.
hubbsii, as the sequence divergence for both markers is above the maximum intraspecies
divergences of ~1% and ~0.5% for each marker, respectively.
Locality and Morphology: MARI-04535 was collected from Fogland Beach South
on
25 July 2023
, and MARI-04594 and MARI-04595 were collected at King’s Beach on
19 September 2023
(Table 7). A few very young vegetative thalli, including MARI-04535,
were found growing on shells and rocks at a depth of about 1.5–2 m in late July; these were
inconspicuous and did not appear to be common (Figure 8A). However, multiple examples
of this species were collected in the drift as epiphytes on coarse algae in September at
King’s Beach, generally not growing to be more than a few centimeters tall (Figure 8B).
Thalli were generally bushy, with axes appearing fuzzy, and unilaterally branched ultimate
branchlets (Figure 8C,E). The ultimate branchlet tips were composed of cylindrical or
swollen, slightly ovoid cells (Figure 8F,G). Every axial cell bore two opposing, distally
arranged branchlets (Figure 8H). Pairs of branchlets were oppositely arranged on the
same axial cell; however, opposite pairs were whorled around the axes, branching in
multiple planes and giving the axes a bushy appearance. Furthermore, basal cells attaching
branchlets to axial cells were globose to spherical and much smaller than the rest of the
branchlet cells (Figure 8I). Rhizoids in the upper thallus developed unilaterally and most
often in triplets from the spherical ultimate branchlet basal cells, similar to Antithamnion
hubbsii (Figure 8J,K). In the lower thallus, basal rhizoids occasionally develop singly from
axial cells or inconspicuous basal branchlet cells (Figure 8L). One specimen collected in
September was tetrasporophytic and bore cruciately divided tetrasporangia borne on short,
multicellular stalks (Figure 8M,N).
Diversity 2024,16, 554 19 of 42
Diversity 2024, 16, x FOR PEER REVIEW 20 of 43
planes and giving the axes a bushy appearance. Furthermore, basal cells aaching branch-
lets to axial cells were globose to spherical and much smaller than the rest of the branchlet
cells (Figure 8I). Rhizoids in the upper thallus developed unilaterally and most often in
triplets from the spherical ultimate branchlet basal cells, similar to Antithamnion hubbsii
(Figure 8J,K). In the lower thallus, basal rhizoids occasionally develop singly from axial
cells or inconspicuous basal branchlet cells (Figure 8L). One specimen collected in Sep-
tember was tetrasporophytic and bore cruciately divided tetrasporangia borne on short,
multicellular stalks (Figure 8M,N).
Figure 7. RAxML phylogenetic trees of Ceramiaceae with Spyridia included as an outgroup. (A)
rbcL-3P. (B) UPA. All unique Ceramiaceae sequences generated in this study are included in these
trees as well as published sequences in GenBank used as context. Sequences generated in this study
start with the five-digit MARI number and end with their GenBank accession numbers; outsourced
sequences do not start with a MARI number.
Figure 7. RAxML phylogenetic trees of Ceramiaceae with Spyridia included as an outgroup. (A)rbcL-
3P. (B) UPA. All unique Ceramiaceae sequences generated in this study are included in these trees as
well as published sequences in GenBank used as context. Sequences generated in this study start with
the five-digit MARI number and end with their GenBank accession numbers; outsourced sequences
do not start with a MARI number.
Remarks: Based on both morphological and molecular results, this species is within
the genus Antithamnion. This species did not match published sequence data of either
species of Antithamnion reported from the NBA (Table 6); however, it groups with various
other Antithamnion in both the rbcL-3P and UPA RAxML trees (Figure 7A,B). Also, this
species shares the following morphological characteristics that place it in this group: basal
branchlet cells smaller than other branchlet cells, at least one order of pinnate branching,
rhizoidal development from basal branchlet cells, and cruciate tetrasporangia borne on
short stalks [
17
,
53
]. Currently, only a few specimens of this species have been collected, and
no older specimens that may be the same species as MARI-04595 have been molecularly
validated from the area; whether this species is native or introduced cannot be assessed.
Diversity 2024,16, 554 20 of 42
What is known, though, is that MARI-04595 is a representative of a species distinct from
other Ceramiaceae historically reported in the NBA and is considered a new report for the
area (Figure 2).
Diversity 2024, 16, x FOR PEER REVIEW 21 of 43
Figure 8. Antithamnion sp. collected from Fogland Beach South and King’s Beach. (A) Habit of thal-
lus in situ, growing on a shell. (B) Habit of thallus in lab. (C,D) Detail of bushy, prolific branching.
(E–G) Detail of branchlet tips, showing cylindrical cells of a vegetative thallus in (E,F), and more
globose cells from a tetrasporophyte in (G). (H) Detail of branchlet near branch apex, showing larger
axial cells with branchlets spreading in multiple planes. (I) Basal cells (arrowheads) of the ultimate
branchlets are globose and smaller than the rest of the branchlet cells; branchlets are whorled around
axial cells. (J) Numerous unpigmented rhizoids extending from a prostrate branchlet. (K) Rhizoids
grow in triplets (arrowhead) from globose basal branchlet cells in the upper thallus. (L) Rhizoids
(arrowheads) grow singly from axial cells in the basal main axes. (M) Tetrasporophyte with many
dark tetraspores interspersed throughout the axes. (N) Cruciately divided tetraspores are borne on
small stalks a few cells tall (arrowhead) on ultimate branchlets.
Remarks: Based on both morphological and molecular results, this species is within
the genus Antithamnion. This species did not match published sequence data of either spe-
cies of Antithamnion reported from the NBA (Table 6); however, it groups with various
other Antithamnion in both the rbcL-3P and UPA RAxML trees (Figure 7A,B). Also, this
species shares the following morphological characteristics that place it in this group: basal
branchlet cells smaller than other branchlet cells, at least one order of pinnate branching,
rhizoidal development from basal branchlet cells, and cruciate tetrasporangia borne on
short stalks [17,53]. Currently, only a few specimens of this species have been collected,
and no older specimens that may be the same species as MARI-04595 have been molecu-
larly validated from the area; whether this species is native or introduced cannot be as-
sessed. What is known, though, is that MARI-04595 is a representative of a species distinct
from other Ceramiaceae historically reported in the NBA and is considered a new report
for the area (Figure 2).
Figure 8. Antithamnion sp. collected from Fogland Beach South and King’s Beach. (A) Habit of thallus
in situ, growing on a shell. (B) Habit of thallus in lab. (C,D) Detail of bushy, prolific branching.
(E–G) Detail of branchlet tips, showing cylindrical cells of a vegetative thallus in (E,F), and more
globose cells from a tetrasporophyte in (G). (H) Detail of branchlet near branch apex, showing larger
axial cells with branchlets spreading in multiple planes. (I) Basal cells (arrowheads) of the ultimate
branchlets are globose and smaller than the rest of the branchlet cells; branchlets are whorled around
axial cells. (J) Numerous unpigmented rhizoids extending from a prostrate branchlet. (K) Rhizoids
grow in triplets (arrowhead) from globose basal branchlet cells in the upper thallus. (L) Rhizoids
(arrowheads) grow singly from axial cells in the basal main axes. (M) Tetrasporophyte with many
dark tetraspores interspersed throughout the axes. (N) Cruciately divided tetraspores are borne on
small stalks a few cells tall (arrowhead) on ultimate branchlets.
Antithamnionella spirographidis (Schiffner) E. M. Wollaston, 1968
Molecular Results: A single specimen, MARI-04622, was collected, and both an rbcL-3P
and UPA sequence were generated for the specimen (Table 5). The rbcL-3P sequence was
a 100% match to A. spirographidis DQ022810 from the Netherlands (Figure 5A), and the
UPA sequence was a 99.19% match to A. ternifolia MK814608 from Spain (Figure 7B). Note
that the UPA sequences of both these species, A. spirographidis MARI-04622, and another
species not discussed in detail in the species treatments, A. floccosa MARI-02643, share
Diversity 2024,16, 554 21 of 42
top BLAST search results and match A. ternifolia MK814608 (Table 1). Both A. floccosa and
A. spirographidis are 0.81% (3bp) divergent from MK814608; however, two of the three
base pair differences are at different locations in the UPA gene between MARI-04622 and
MARI-02643. Even though MARI-04622 and MARI-02643 are the closest neighbors to each
other out of the species we collected, they are 1.35% divergent from one another; they
are further apart from each other than from MK814608 (Figure 7B). The nearest neighbor
based on the rbcL-3P sequences was the other Antithamnion sp. (Species Treatment II),
which differed from MARI-04622 by 9.74–9.81%. However, the large interspecies gap in
the rbcL-3P data that is not seen in the UPA data is likely because we do not have rbcL-3P
sequence data for MARI-02643 or other potentially closer-related species.
Locality and Morphology: The single specimen MARI-04622 was found floating in
shallow waters off Lighthouse Beach, Chatham, MA, on 29 October 2023 (Table 7; Figure 9A).
The thallus was light pink, uniseriate throughout, and had adventitious branchlets on every
axial cell at the base of the thallus (Figure 9B). Branching was somewhat planar and in two
orders: alternate then pinnate; however, sometimes branchlets would not be paired near
branchlet tips and would be unilaterally arranged or paired branchlets were of unequal
lengths (Figure 9C–F). In the lower axes, 2–3 ultimate branchlets are whorled and situated
distally on axial cells. These branchlets often have one to a few gland cells growing
adaxially on cells near the bases of the branchlets (Figure 9G,H). Tetrahedrally divided,
sessile tetrasporangia are present in the upper axes, growing singularly and adaxially on
basal cells of ultimate branchlets (Figure 9I–L).
Remarks: The specimen collected from Chatham, MA, is a 100% match (rbcL-3P) and
is a strong morphological match to Antithamnionella spirographidis [
42
,
54
]. However, MARI-
04622 also matches the description of Scagelia americana, as reported from the NBA [
17
]. The
description for A. spirographidis, as given by [
42
,
54
], is nearly identical to that of S. americana
in Mathieson & Dawes [
17
]. All three described a species with irregular to alternate
branching, with every axial cell bearing 2–4 whorled branchlets, branchlet pairs having
uneven lengths, apical cells with pointed tips, abundant gland cells growing adaxially on
basal branchlet cells, and tetrasporangia being sessile, oblong, cruciately divided, and near
the bases of branchlets. However, while S. americana is still a valid name [
16
], it was listed
as a heterotypic synonym of Scagelia pylaisaei by Bruce [
42
]. S. pylaisaei was reported to be a
highly morphologically variable species, and there was also high molecular diversity within
the group that was inconsistent with morphologies, which was presumed to account for the
confusion in species determinations of Scagelia in Canadian waters [
42
]. Therefore, since
the identity of S. americana is ambiguous and the description of S. americana in Mathieson
& Dawes [
17
] is nearly identical to the less ambiguously described A. spirographidis, the
description of S. americana may refer to a misidentified A. spirographidis. If this is true, then
A. spirographidis may have been present in the NBA for some time; however, this cannot
be confirmed without assessing older material. Regardless of what species Mathieson &
Dawes [
17
] were referring to, based on the morphological and molecular data, MARI-04622
is determined to be A. spirographidis, and this taxon is reported from the western Atlantic
for the first time (Figure 1). A single collection of this species, especially one that was
not found to be actively growing, is not enough to determine whether this species has
established a population in the area, but this species should be monitored. The origin of
A. spirographidis is somewhat disputed; however, it is likely native to the Pacific [
55
]. It is
found throughout the Pacific, including the west coast of the United States and Canada,
Japan, Russia, China, and Australia, as well as much of the Mediterranean [
16
]. Due to its
widespread distribution in the Pacific, it is believed that this species was introduced to the
Mediterranean some time before the 1970s, likely through hull fouling or contaminated
ballast water [56,57].
Diversity 2024,16, 554 22 of 42
Diversity 2024, 16, x FOR PEER REVIEW 23 of 43
Mediterranean [16]. Due to its widespread distribution in the Pacific, it is believed that
this species was introduced to the Mediterranean some time before the 1970s, likely
through hull fouling or contaminated ballast water [56,57].
Figure 9. Antithamnionella spirographidis collected from Lighthouse Beach, Chatham, MA. (A) Full
specimen. (B) Lower axes. (C–E) Detail of axes in two orders of branching, alternate and oppo-
site/pinnate. (F) Axis termini end in a slightly sinusoidal arrangement (black line). (G,H) Detail of
lower axes with whorled branchlets. Gland cells (arrowheads) present and adaxial on whorled
branchlets near the bases of branchlets. (I–K) Numerous developing tetrasporangia are present,
growing singularly, sessile, and adaxial on basal cells of ultimate branchlets. (L) Detail of tetrahe-
drally divided tetrasporangium.
Dasya elegans (G. Martens) C. Agardh
Molecular Results: Both rbcL-3P and UPA sequences were obtained from two speci-
mens, MARI-04392 and MARI-04407. The rbcL-3P sequences were a 100% match to Dasya
sp. 1 baillouviana MW698713 from Nova Scotia as well as D. “baillouviana” FM993088 from
The Netherlands (Figure 5A). D. “baillouviana” FM993088 separates from MARI-04392 in
Figure 9. Antithamnionella spirographidis collected from Lighthouse Beach, Chatham, MA. (A) Full
specimen. (B) Lower axes. (C–E) Detail of axes in two orders of branching, alternate and oppo-
site/pinnate. (F) Axis termini end in a slightly sinusoidal arrangement (black line). (G,H) Detail
of lower axes with whorled branchlets. Gland cells (arrowheads) present and adaxial on whorled
branchlets near the bases of branchlets. (I–K) Numerous developing tetrasporangia are present,
growing singularly, sessile, and adaxial on basal cells of ultimate branchlets. (L) Detail of tetrahedrally
divided tetrasporangium.
Dasya elegans (G. Martens) C. Agardh
Molecular Results: Both rbcL-3P and UPA sequences were obtained from two speci-
mens, MARI-04392 and MARI-04407. The rbcL-3P sequences were a 100% match to Dasya
sp. 1 baillouviana MW698713 from Nova Scotia as well as D. “baillouviana” FM993088 from
The Netherlands (Figure 5A). D. “baillouviana” FM993088 separates from MARI-04392 in the
rbcL-3P RAxML tree because the sequences do not completely overlap (each is a sequence
from slightly different segments of the rbcL gene, Figure 5A). Both UPA sequence BLAST
search results were a 98.64% match to Dasya sp. HQ421299 from Hawaii, and a 98.37%
Diversity 2024,16, 554 23 of 42
match to D. baillouviana HQ421392, also from Hawaii (Figure 5B). Intraspecies divergences
were 0% for both markers (Table 6). The nearest neighbor we collected was D. pedicellata
according to both markers, and this species was 1.00–1.16% and 1.63% divergent from D.
pedicellata for rbcL-3P and UPA, respectively.
Locality and Morphology: Two specimens were collected from the estuarine RWU
waterfront, one on 24 May 2023, and the other on 4 June 2023. Both specimens were
lithophytes in the shallow subtidal, and they were abundant during late spring/early
summer at this site (Table 7). Thalli were light, peach pink to deep red, with main axes
that were covered in adventitious, pigmented, uniseriate branchlets that gave the thallus
a fuzzy appearance (Figure 10A–E). Often, branching was rather sparse, and there was a
discernible central axis from which laterals extended almost perpendicularly (Figure 10A).
In cross-section, the main axes had 5–6 pericentral cells (Figure 10F,G). The main axes were
heavily corticated (Figure 10H). Adventitious branchlets were composed of often long,
cylindrical cells, and branchlets divided dichotomously, bearing tetrasporangial stichidia
on short multicellular stalks (Figure 10I–K). This species is almost inseparable from D.
pedicellata based on morphology alone in the NBA; however, D. elegans had much sparser
lateral branches from the main axes, and adventitious branchlets were not as abundant and
did not obscure the main axes as much as in D. pedicellata. However, aside from the fact
that these characters are relatively subjective, since very few specimens of D. elegans were
examined in this study, these characters may also not be universal.
Remarks: Only one species of Dasya has previously been reported from the NBA,
and that was D. baillouviana, which is now considered an invalid name due to the lack of
any surviving type material and the ambiguity of the identity of the type specimen [
58
].
Following the rejection of the name D. baillouviana,D. pedicellata was promoted back to
full species status and has been applied as the name for Dasya collected in the NBA since
the type specimen of D. pedicellata was from New York [
58
]. However, two genotypes of
Dasya were recovered in this study and these were named D. elegans and D. pedicellata
(Section Dasya pedicellata (C. Agardh) C. Agardh, 1824 Dasya pedicellata). Although both
recovered species were very similar morphologically, molecular data suggest that these
are different species, since the minimum sequence divergence between D. elegans and D.
pedicellata (1.00% and 1.63% for rbcL-3P and UPA, respectively) were both greater than their
intraspecies divergences of 0% for both markers. Therefore, we report two species of Dasya
from the NBA. The historical reports of only a single species of Dasya from the NBA were
likely a result of the nearly identical morphologies of the two molecularly validated species,
and/or potentially because D. elegans was introduced recently to the Atlantic Northwest
from the Eastern Atlantic, since our sequence data matches some from the Netherlands
(D. “baillouviana” FM993088). The species represented by MARI-04392 and MARI-04407,
which is molecularly distinct from the multiple specimens of D. pedicellata that were also
collected in this study, is provisionally named D. elegans.D. elegans is a name still considered
synonymous to D. pedicellata, but it is currently being considered to be resurrected for this
second species [
16
] (G.W. Saunders, pers. comm.). The type specimen of D. elegans is from
Italy, which suggests that if MARI-04392 and MARI-04407, collected in the NBA, are the
same species as the Italian holotype, then this would support the idea that this species was
introduced from the Mediterranean, as mentioned above. More taxonomic work is needed
in this genus to fully understand the origins and distributions of the constituents of this
genus, as many species are morphologically challenging to separate; however, this is a
new report from the NBA considering that only one species of Dasya has been reported
historically from the area (Figure 2).
Diversity 2024,16, 554 24 of 42
Diversity 2024, 16, x FOR PEER REVIEW 25 of 43
Figure 10. Dasya cf. elegans collected from the RWU waterfront, Bristol, RI. (A,B) General habit of
thallus in lab. (C–E) Detail of axes with relatively sparse adventitious branchlets that give axes a
fuzzy appearance, but by no means obscure the main axes. (F,G) Cross-sections of axes near branch
termini, showing heavy cortication and six and five pericentral cells (numbered) in (F,G), respec-
tively. (H) Surface view of the corticated main axis, devoid of adventitious branchlets. (I,J) Detail of
uniseriate adventitious branchlets, which in (J) can be seen dividing dichotomously. (K) Tetraspo-
rangial stichidia are borne on short stalks of a few cells off the adventitious branchlets.
Streblocladieae sp.
Molecular Results: Both rbcL-3P and UPA sequences were generated for a single spec-
imen, MARI-04466 (Table 5). Neither marker produced a very close BLAST search result
with any published sequences, indicating that there are no published sequence data for
the species of MARI-04466 (Table 6, Figures 5A and 6B). The rbcL-3P sequence was closest
to Kapraunia pentamera HM573564 from Panama, which was only a 93.30% match (Figure
5A), and the UPA sequence was a 97.83% match to Polysiphonia sp. HQ421052 from Hawaii.
The nearest molecular neighbor we collected according to the rbcL-3P markers was
Kapraunia schneideri, which was 7.26–7.94% divergent from MARI-04466. Acanthosiphonia
echinata was the nearest molecular neighbor according to the UPA marker, which was 3.78%
divergent from MARI-04466.
Figure 10. Dasya cf. elegans collected from the RWU waterfront, Bristol, RI. (A,B) General habit of
thallus in lab. (C–E) Detail of axes with relatively sparse adventitious branchlets that give axes a
fuzzy appearance, but by no means obscure the main axes. (F,G) Cross-sections of axes near branch
termini, showing heavy cortication and six and five pericentral cells (numbered) in (F,G), respectively.
(H) Surface view of the corticated main axis, devoid of adventitious branchlets. (I,J) Detail of uniseri-
ate adventitious branchlets, which in (J) can be seen dividing dichotomously. (K) Tetrasporangial
stichidia are borne on short stalks of a few cells off the adventitious branchlets.
Streblocladieae sp.
Molecular Results: Both rbcL-3P and UPA sequences were generated for a single
specimen, MARI-04466 (Table 5). Neither marker produced a very close BLAST search
result with any published sequences, indicating that there are no published sequence data
for the species of MARI-04466 (Table 6, Figures 5A and 6B). The rbcL-3P sequence was
closest to Kapraunia pentamera HM573564 from Panama, which was only a 93.30% match
(Figure 5A), and the UPA sequence was a 97.83% match to Polysiphonia sp. HQ421052 from
Hawaii. The nearest molecular neighbor we collected according to the rbcL-3P markers was
Kapraunia schneideri, which was 7.26–7.94% divergent from MARI-04466. Acanthosiphonia
echinata was the nearest molecular neighbor according to the UPA marker, which was 3.78%
divergent from MARI-04466.
Diversity 2024,16, 554 25 of 42
Locality and Morphology: MARI-04466 was collected from the southwestern end of
Ninigret Pond by the East Beach boat launch in shallows less than one meter deep on
21 June 2023
(Table 7). During a snorkel that lasted over 30 min, only the one specimen was
collected, growing as a large (~11 cm tall) but inconspicuous lithophyte that looked and felt
very similar to a lot of brown and green algal growth in the same area. The thallus was dull
orangish brown and had very thin branches that were not easy to make out with the naked
eye (Figure 11A). Branching was alternate to irregular throughout, with central, uncurved
axes that bore adventitious branchlets. Branching was abundant and followed the echinate
branching patterns of some members of the tribe Streblocladieae (Figure 11B,C).
Diversity 2024, 16, x FOR PEER REVIEW 26 of 43
Locality and Morphology: MARI-04466 was collected from the southwestern end of
Ninigret Pond by the East Beach boat launch in shallows less than one meter deep on 21
June 2023 (Table 7). During a snorkel that lasted over 30 minutes, only the one specimen
was collected, growing as a large (~11 cm tall) but inconspicuous lithophyte that looked
and felt very similar to a lot of brown and green algal growth in the same area. The thallus
was dull orangish brown and had very thin branches that were not easy to make out with
the naked eye (Figure 11A). Branching was alternate to irregular throughout, with central,
uncurved axes that bore adventitious branchlets. Branching was abundant and followed
the echinate branching paerns of some members of the tribe Streblocladieae (Figure
11B,C).
Figure 11. Unidentified species of Streblocladieae collected from Ninigret Pond. (A) Habit of thallus
in lab. (B) Detail of branching paern of herbarium press. (C–E) Detail of axes in surface view.
Branchlets arise from nodes between cells and stem from the central axis. (F) Cross-section showing
four pericentral cells (numbered). (G) The branchlet tip has a few trichoblasts and an exposed, sin-
gular apical cell. (H) Rhizoid has an open connection to the axial cell (arrowhead). (I) Three large
rhizoids (arrowheads) found at the base of the thallus. (J,K) Branchlets are swollen with tetraspo-
rangia in a slightly spiral series (arrowheads). (L) A tetrahedrally divided tetrasporangium. Note:
Images of (H,I,L) were taken after rehydrating material from the herbarium specimen.
Figure 11. Unidentified species of Streblocladieae collected from Ninigret Pond. (A) Habit of thallus
in lab. (B) Detail of branching pattern of herbarium press. (C–E) Detail of axes in surface view.
Branchlets arise from nodes between cells and stem from the central axis. (F) Cross-section showing
four pericentral cells (numbered). (G) The branchlet tip has a few trichoblasts and an exposed,
singular apical cell. (H) Rhizoid has an open connection to the axial cell (arrowhead). (I) Three
large rhizoids (arrowheads) found at the base of the thallus. (J,K) Branchlets are swollen with
tetrasporangia in a slightly spiral series (arrowheads). (L) A tetrahedrally divided tetrasporangium.
Note: Images of (H,I,L) were taken after rehydrating material from the herbarium specimen.
Axes were ecorticate throughout and had four pericentral cells (Figure 11D–F). Tri-
choblasts were present around branchlet apices, and a single apical cell was often clearly
visible at branch termini (Figure 11G). Rhizoids were connected to axial cells via an open
Diversity 2024,16, 554 26 of 42
connection and were abundant on the basal growth of the thallus (Figure 11H,I). MARI-
04466 was a tetrasporophyte, whose tetrasporangia were arranged in spiral series near
branch apices, causing branchlets to swell and warp (Figure 11J). Sometimes, the spiral
character is not very pronounced in some branchlets (Figure 11K). Tetrasporangia were
tetrahedrally divided (Figure 11L). The morphology of MARI-04466 loosely matches the
description of Bryocladia subtilissima (previously Polysiphonia subtilissima), which has been
reported from the NBA by multiple sources [
17
,
34
,
35
]. B. subtilissima is an euryhaline
species that can grow in both freshwater and marine environments and is often found
in estuarine/brackish waters, is ecorticate, has four pericentral cells throughout, and has
unicellular rhizoids with an open connection to the parent axial cells [
17
,
59
]. However,
MARI-04466 differed in at least one major way from B. subtilissima, which is known to be a
characteristic used for separating species in this group. Tetrasporangia were arranged in
spiral series in MARI-04466, whereas tetrasporangia were arranged in straight series in B.
subtilissima [17].
Remarks: Considering the molecular results indicating that this species does not
match any published sequence data, and that this species does not match any species
historically reported from the area morphologically, this species appears to be either an
undescribed species or potentially an introduced species of obscure origin for which no
sequence data is currently available. There is another possibility that seems to be the more
likely situation. As mentioned above, this species would key out to B. subtilissima, which is
a species reported from the NBA and which we did not otherwise collect. However, MARI-
04466 is almost certainly not B. subtilissima, since our specimen differs morphologically
from B. subtilissima since the tetrasporangia are arranged differently, as discussed above.
Furthermore, the molecular results agree that these are not the same species, since there
are published sequence data for B. subtilissima, which MARI-04466 does not match well
with at all. The closest published B. subtilissima sequence was B. subtilissima (as Polysiphonia
subtilissima) JX294918 from Spain, which was only an 86.85% match to the rbcL-3P sequence
for MARI-04466 (Figure 5A). However, there is the possibility that specimens of the species
as MARI-04466 were collected in the NBA and were mistakenly identified as B. subtilissima,
just as the case was with Chondria atropurpurea being called Chondria capillaris and Dasya
pedicellata being called Dasya baillouviana. Further collections of the species of MARI-04466
and comparison to historical collections from the NBA may help clarify the status of this
species. What is clear, though, is that this species is not Bryocladia subtilissima and is
considered a new report for the region (Figure 2).
3.4.2. Species with Taxonomic Ambiguities That Are Not New Reports: (6 Species)
Antithamnion hubbsii E. Y. Dawson
Molecular Results: Sequences were obtained from seven specimens, four of which
both rbcL-3P and UPA sequences were generated for. The rbcL-3P sequences were a 100%
match to both A. nipponicum AY591928 and A. hubbsii KJ202093, both specimens from
North Carolina, as well as a 99.85% match to three more A. nipponicum and one A. hubbsii
from various other localities (Figure 7A). UPA sequences were a 100% match to A. hubbsii
KJ202103 from North Carolina (Figure 7B). Intraspecies divergence was 0% for both markers
(Table 6). The closest neighbor to A. hubbsii for both markers was Antithamnion sp. (Species
Treatment II), from which A. hubbsii sequences differed by 6.15–6.30% and 1.63% for rbcL-3P
and UPA, respectively.
Locality and Morphology: Sequenced specimens were collected from Black Point,
King’s Beach, and Fort Wetherill from June, July, and September (Table 7). Specimens were
collected frequently on various coarse algae in high wave action zones along the open coast
from July through November. In most cases, thalli were gregarious and created spreading,
noticeable, epiphytic turfs on the distal, wave-exposed growth of subtidal algae, most
commonly Chondrus (Figure 12A–C). Tetrasporophytes were observed in late July; however,
none of this material was imaged or sequenced. Thalli were uniseriate throughout, and
while specimens had decussate branching patterns (Figure 12D), most specimens had
Diversity 2024,16, 554 27 of 42
strictly planar pinnate branching in two orders (Figure 12E–H). Branchlets were connected
to main axes by small spherical basal cells smaller than the rest of the branchlet cells,
gland cells were common (Figure 12I–K), occasionally more than 5 per branchlet, and
rhizoids generally grew in triplets from the spherical branchlet basal cells (Figure 12K,L).
The uniseriate thallus structure, planar pinnate branching patterns, and small and spherical
basal branchlet cells are distinctive characteristics that are consistent with the morphology
described for A. hubbsii [17,53].
Diversity 2024, 16, x FOR PEER REVIEW 29 of 43
hubbsii had the decussate branching paern that is supposed to be unique to A. cruciatum
[17].
Figure 12. Antithamnion hubbsii collected from Black Point, King’s Beach, and Fort Wetherill. (A)
General habit in situ. (B,C) Habit of thallus. (D) Detail of non-planar, slightly decussate branching
that is atypical of the species MARI-04431. (E–G) Detail of typical planar, pinnate branching paern.
(H) Lateral view showing the planar structure of two fronds. (I) Detail of branchlet with four adaxial
gland cells (arrowheads) on the basal ultimate branchlet cells. (J,K) Detail of branchlets along axial
cells. Note that the basal branchlet cells (arrowheads) are globose and smaller than the rest of the
branchlet cells. In (K), there are two clear rhizoids (arrows) growing from the central, lower-right
basal branchlet cell. (L) Multiple bundles of unpigmented rhizoids extending up and to the left from
the prostrate axis.
Ceramium facetum G.W. Saunders & C. W. Schneider, 2024
Molecular Results: A total of 13 specimens were sequenced, and for 8 of these, both
rbcL-3P and UPA sequences were generated. The rbcL-3P sequences were a 100% match
to C. facetum (Ceramium sp.1GoSL) in the Barcode of Life Data System (BOLDS; [65]) and a
100% match to C. diaphanum KF367765 from North Carolina in GenBank (Figure 7A). The
UPA sequences were also a 100% match to the same GenBank specimen as rbcL-3P, C.
Figure 12. Antithamnion hubbsii collected from Black Point, King’s Beach, and Fort Wetherill. (A) Gen-
eral habit in situ. (B,C) Habit of thallus. (D) Detail of non-planar, slightly decussate branching that
is atypical of the species MARI-04431. (E–G) Detail of typical planar, pinnate branching pattern.
(H) Lateral view showing the planar structure of two fronds. (I) Detail of branchlet with four adaxial
gland cells (arrowheads) on the basal ultimate branchlet cells. (J,K) Detail of branchlets along axial
cells. Note that the basal branchlet cells (arrowheads) are globose and smaller than the rest of the
branchlet cells. In (K), there are two clear rhizoids (arrows) growing from the central, lower-right
basal branchlet cell. (L) Multiple bundles of unpigmented rhizoids extending up and to the left from
the prostrate axis.
Diversity 2024,16, 554 28 of 42
Remarks: The name Antithamnion hubbsii has a complicated taxonomic history. A.
hubbsii was first described from Baja California, Mexico, in 1962 and has since been reported
widely across the globe [
53
]. However, this species has long been confused and used
almost interchangeably with A. pectinatum and A. nipponicum [
53
]. A. pectinatum has been
identified as a species that was introduced to the Mediterranean [
60
,
61
]; however, it was
determined that these specimens thought to be A. pectinatum should be A. hubbsii [
62
].
Furthermore, A. nipponicum, which has been recorded from the Western Pacific and has also
been reported to have been introduced in the Mediterranean [
63
] and North Carolina [
53
],
has since been synonymized with A. pectinatum, whose type locality is in New Zealand [
62
].
In short, all specimens that were called A. hubbsii,A. nipponicum, or A. pectinatum outside
of New Zealand have been renamed A. hubbsii.A. nipponicum is no longer a valid name,
and A. pectinatum is only present in New Zealand. However, no A. hubbsii from the type
locality of Baja California have been sequenced, so there is a chance that molecular A.
hubbsii recorded from the Mediterranean, western Pacific, and western Atlantic are not the
same as true A. hubbsii. For the purpose of this study, the name A. hubbsii was maintained
for specimens collected and sequenced in the NBA. Five of the seven specimens collected
in this study that were a molecular match to North Carolinian Antithamnion hubbsii were
also a morphological match to this species, as described above. Furthermore, both UPA
and rbcL-3P sequences were generated for three of these five specimens, and molecular
species determinations for both markers for each of the three specimens agreed with A.
hubbsii. Furthermore, A. hubbsii has been reported from the NBA [
17
], and it was first
reported to have been introduced to Connecticut in 1991 under the name Antithamnion
cf. nipponicum [
64
]. Therefore, since the morphology and molecular data agree for these
specimens as well as with historical reports, the current presence and identity of A. hubbsii
is reaffirmed in the NBA. However, two samples from Fort Wetherill and one from King’s
Beach did not fit the above morphological descriptions for A. hubbsii, but were molecular
matches to A. hubbsii (for those that were barcoded). These included MARI-04431, for
which a UPA sequence was generated; MARI-04476, for which we did not have any
sequence data; and MARI-04560, for which an rbcL-3P sequence was generated. Each of
these specimens had decussate branching patterns (i.e., branching in two more or less
perpendicular planes, Figure 12D). While MARI-04560 was found as a turf on Chondrus-like
typical A. hubbsii specimens, MARI-04431 and MARI-04476 were found to be growing in
sparse patches along more sheltered, lower axes of coarse algae. There are two species
of Antithamnion reported from the NBA—A. hubbsii and A. cruciatum—and MARI-04431,
MARI-04476, and MARI-04560 agree morphologically with A. cruciatum.A. hubbsii has
been described as having a planar branching pattern, while A. cruciatum has a decussate
branching pattern [
17
]. However, the UPA sequence for MARI-04431 and the rbcL-3P
sequence for MARI-04560 are matches to other A. hubbsii sequences we generated, and the
A. hubbsii rbcL-3P sequences were distinct from A. cruciatum AY136277 (Figure 7A). This,
along with the fact that sequences were 0% divergent from one another for both markers,
suggests that there may be only one species of Antithamnion present in the NBA, or if A.
cruciatum is also present and it was not encountered by this study, then the two species
cannot be separated by their branching pattern, as multiple specimens that barcoded
to A. hubbsii had the decussate branching pattern that is supposed to be unique to A.
cruciatum [17].
Ceramium facetum G.W. Saunders & C. W. Schneider, 2024
Molecular Results: A total of 13 specimens were sequenced, and for 8 of these, both
rbcL-3P and UPA sequences were generated. The rbcL-3P sequences were a 100% match
to C. facetum (Ceramium sp.1GoSL) in the Barcode of Life Data System (BOLDS; [
65
]) and
a 100% match to C. diaphanum KF367765 from North Carolina in GenBank (Figure 7A).
The UPA sequences were also a 100% match to the same GenBank specimen as rbcL-3P, C.
diaphanum KF367775 (Figure 7B). The intraspecies divergence for rbcL-3P and UPA was
0–0.07% and 0–0.28%, respectively (Table 6). The nearest neighbor we collected to this
Diversity 2024,16, 554 29 of 42
species was C. virgatum for rbcL-3P, which differed from C. facetum by 6.78–7.62%, and the
nearest neighbor according to our UPA sequence data was C. secundatum, which differed
from our sequences by 1.08–1.41%.
Locality and Morphology: Specimens were collected throughout the summer from
late May to mid-August in a variety of habitats, from protected estuaries to salt marsh
headwaters to tidepools and drift material on the open coast (Table 7). This was most
commonly collected as a lithophyte forming dense, bushy mats on rocks in the low intertidal
zone in more protected areas but also occasionally collected as an epiphyte on coarser algae.
Thalli were incompletely corticated, profusely branched, and often strictly dichotomously
branched (Figure 13A–D). Branchlet tips were recurved and pincer-shaped, a characteristic
typical of the genus Ceramium (Figure 13E). Branching dichotomies occurred at the nodes
(Figure 13F). Branchlet tips were recurved and pincer-shaped, a characteristic typical of
the genus Ceramium (Figure 13E). In cross-section, nodes had 6–7 large, round periaxial
cells that were distinct from cortical cells, which separates this species from the only
other incompletely corticated ceramiacean alga reported from the area, Ceramothamnion
translucidum, whose periaxial cells are fewer (~5) and are similar in size to cortical cells
(Figure 13G,H). Sun-bleached thalli from much calmer, protected waters appear much paler
than thalli from high wave action or shaded areas (Figure 13H). In some thalli, adventitious
branchlets were present, but were still mostly dichotomously branched with pincer-shaped
tips (Figure 13I,J). Tetrasporophytes were collected in early June and had tetrasporangia
organized in whorls around the nodes (Figure 13L,M). Cystocarpic thalli were collected in
late July, and pairs of gonimolobes developed at branching dichotomies (Figure 13N).
Remarks: This species has long been reported from the NBA as C. diaphanum, whose
holotype is from Scotland. However, routine DNA barcoding of specimens from the
Northwest Atlantic has revealed that the incompletely corticated Ceramium that has long
been identified as C. diaphanum in the northwest Atlantic is distinct from the true C.
diaphanum from the eastern Atlantic [
36
]. The northwestern Atlantic species has just recently
been described as a new species, C. facetum [
66
]. The sequences generated in this study
are consistent with this finding and match “C. diaphanum” from the northwestern Atlantic,
not Europe. A specimen from Cornwall, England, published as C. diaphanum FR871401, is
only a 94.66% match to the rbcL-3P sequences we generated and is the closest molecular
match of any published European C. diaphanum sequences (Figure 7A). Considering both
the morphology discussed above and the molecular results, C. facetum is regarded as
established in the NBA and has been mistaken for an extralimital but morphologically
similar European species.
Ceramium plenatunicum G. W. Saunders & C. W. Schneider, 2024
Molecular Results: For three different specimens, rbcL-3P sequences were obtained;
however, no complete UPA sequences were generated. The rbcL-3P sequences were a 100%
match to C. plenatunicum (Ceramium sp. 2virgatum) GWS006236 from New Brunswick in
BOLDS, and a 98.80% match to C. derbesii FR775779 from Italy in GenBank (Figure 7A).
The intraspecies divergence of the three sequences was 0% (Table 6). The nearest collected
neighbor was C. secundatum, which differed from this species by 2.54–3.63%.
Locality and Morphology: Two specimens were collected from the protected, estuarine
north shore of Fogland Beach on 25 July 2023 (Table 7). An rbcL-3P sequence from another
herbarium specimen was also obtained, but the collection data for that specimen are
not included here. The summer 2023 specimens were abundant epiphytes in shallow,
<20 cm deep water at low tide, often growing on gracilariacean algae and mixed with
Chondria baileyana (Figure 14A–C). Branching was strictly dichotomous with occasional
adventitious branches, and branching angles were wide, spreading, and often >60
◦
except
at the branchlet tips (Figure 14D). Thalli were completely corticated throughout but may
still appear banded, as the nodes were sometimes darker than internodes (Figure 14E–G).
In the cross-section, nodes had 6–7 circular periaxial cells (Figure 14H). Branchlet tips were
Diversity 2024,16, 554 30 of 42
typical for the genus Ceramium, with pincer-shaped termini that were slightly inrolled
(Figure 14I). No reproductive material was collected.
Diversity 2024, 16, x FOR PEER REVIEW 31 of 43
Figure 13. Ceramium facetum collected from a variety of estuarine habitats throughout Rhode Island.
(A) Darker thallus collected in murky rapids at the head of Bristol Harbor, RI. (B,C) Detail of dichot-
omous branching paern. (D–F) Axes are incompletely corticated throughout and end in recurved,
pincer-shaped tips. (G) Cross-section of node revealing six periaxial cells (numbered). (H) Lighter
thallus collected in calm clear waters where there is high light exposure on the north side of Fogland
Beach, Tiverton, RI. (I) Detail of branching paern, which is mostly dichotomous but has numerous
adventitious branchlets (arrowheads) arising from nodes. (J) Branchlet tips are recurved. (K) Cross-
section of node revealing seven periaxial cells (numbered). (L,M) Emergent tetrasporangia (arrow-
heads) organized around nodes. (N) Dark, paired gonimolobes growing at the branch node.
Ceramium plenatunicum G. W. Saunders & C. W. Schneider, 2024
Molecular Results: For three different specimens, rbcL-3P sequences were obtained;
however, no complete UPA sequences were generated. The rbcL-3P sequences were a 100%
match to C. plenatunicum (Ceramium sp. 2virgatum) GWS006236 from New Brunswick in
BOLDS, and a 98.80% match to C. derbesii FR775779 from Italy in GenBank (Figure 7A).
The intraspecies divergence of the three sequences was 0% (Table 6). The nearest collected
neighbor was C. secundatum, which differed from this species by 2.54–3.63%.
Figure 13. Ceramium facetum collected from a variety of estuarine habitats throughout Rhode Island.
(A) Darker thallus collected in murky rapids at the head of Bristol Harbor, RI. (B,C) Detail of
dichotomous branching pattern. (D–F) Axes are incompletely corticated throughout and end in
recurved, pincer-shaped tips. (G) Cross-section of node revealing six periaxial cells (numbered).
(H) Lighter thallus collected in calm clear waters where there is high light exposure on the north side
of Fogland Beach, Tiverton, RI. (I) Detail of branching pattern, which is mostly dichotomous but has
numerous adventitious branchlets (arrowheads) arising from nodes. (J) Branchlet tips are recurved.
(K) Cross-section of node revealing seven periaxial cells (numbered). (L,M) Emergent tetrasporangia
(arrowheads) organized around nodes. (N) Dark, paired gonimolobes growing at the branch node.
Diversity 2024,16, 554 31 of 42
Diversity 2024, 16, x FOR PEER REVIEW 33 of 43
Branching was strictly dichotomous, and branches had acute angles of around 60° or less,
and axes were incompletely corticated throughout (Figure 15C–E). The darker, central ax-
ial filament could often be seen from the surface view of newer axes near branchlet tips
(Figure 15F). In cross-section, nodes had about 5 periaxial cells that were slightly larger
than the abundant, surrounding cortical cells (Figure 15G). Periaxial cells were generally
small enough so that it was possible to take a cross-section at a node that did not include
the periaxial cells and only revealed cortical cells (Figure 15H). The branchlet tips were
often slightly inrolled and pincer-shaped (Figure 15I–K); however, sometimes, the branch-
let tips were straight and not recurved at all (Figure 15L). These characteristics are con-
sistent with the original description of Ceramothamnion translucidum [36]. Furthermore, the
relatively smaller size of periaxial cells in Ceramothamnion translucidum separates it from
species of the genus Ceramium, whose periaxial cells are often more numerous and are
generally much larger and distinct from cortical cells at nodes [67].
Figure 14. Ceramium plenatunicum collected from Fogland Beach, Tiverton, RI. (A,B) General habit of
thallus in situ. (C) Thallus in the lab. (D) Detail of wide-angled, spreading dichotomous branching
pattern. (E,F) Axes are corticated throughout but may still appear banded, as cortical cells at nodes
appear as dark bands in surface view. (G) Branching is dichotomous throughout. (H) Cross-section at
node revealing seven circular periaxial cells (numbered). (I) Branchlet tips are not strongly recurved
in these thalli, but they are still pincer-shaped.
Remarks: Historically, there have been two completely corticated species of Ceramium
reported from the NBA. These would be C. secundatum and C. virgatum, both of which
were recovered in this study, and we have multiple rbcL-3P and UPA sequences that agree
with the morphological identifications confirming these identities (Table 1). However, a
third species of completely corticated Ceramium was recovered as well, and that species
both matches the morphology and barcode of C. plenatunicum, which was only recently
Diversity 2024,16, 554 32 of 42
described based on specimens collected in New Brunswick [
36
]. This species has also
been genetically verified in Rhode Island from an epiphyte on Grateloupia [
36
]. This
species mainly differs from the other two completely corticated Ceramium because it has
much wider branching dichotomies, but there may be other differences that would be
better elucidated by collection/study of more specimens. One such potential difference
is how the periaxial cells of C. plenatunicum seem to be much more circular than those
of C. secundatum and virgatum, whose periaxial cells are often slightly oblong or sector-
shaped. Since this species is morphologically very similar to the other completely corticated
Ceramium reported from the NBA, it is likely that this species has long been overlooked and
is probably not introduced to the area; but the origin and history of this species is uncertain
in the NBA. However, considering that C. plenatunicum has been molecularly verified from
the NBA by another lab [66], it is not included as a new report for the area (Figure 2).
Ceramothamnion translucidum G. W. Saunders & C. W. Schneider, 2024
Molecular Results: Sequences for five specimens were obtained, and for three of these
specimens, both rbcL-3P and UPA sequences were generated. The rbcL-3P sequences were a
100% match to “Ceramium sp. 2 DWF-2013” KF367768 from North Carolina (Figure 7A). UPA
sequences were also a 100% match to the same species from the same locality, “Ceramium sp.
2 DWF-2013,” from North Carolina (Figure 7B). These species, “Ceramium sp. 2 DWF-2013,”
are matches to holotype sequences of Ceramothamnion translucidum [
36
], so the species
validated in this study is also therefore C. translucidum. Intraspecies divergence for the
rbcL-3P and UPA sequences generated were 0–0.15% and 0%, respectively (Table 6). The
nearest neighbor we collected, as indicated by both rbcL-3P and UPA sequence data, was
C. virgatum, which this species differed from by 8.57–9.54% and 5.41% for rbcL-3P and
UPA, respectively.
Locality and Morphology: Four specimens from three different sites at Ninigret Pond
and one specimen from Horseneck Beach were collected in June and July of 2023 (Table 7).
A fifth specimen, an older herbarium specimen, MARI-03342, collected from Sakonnet
Point in September 1972, was also barcoded to this species. This specimen was described
as an epiphyte on Fucus and had an annotation and drawing that indicated that it was
tetrasporophytic, with tetrahedrally divided tetraspores growing emergent from axial
nodes. All specimens collected in the summer of 2023 were nonreproductive. Thalli
were generally in small, epiphytic tangled clusters no more than a few centimeters across,
collected in shallow subtidal (<1 m depth) at Ninigret Pond (Figure 15A,B). The specimen
from Horseneck Beach was a small tuft growing on Ascophyllum, collected in the drift. The
older specimen was much larger and spreading, a dense mat over 10 centimeters across.
Branching was strictly dichotomous, and branches had acute angles of around 60◦or less,
and axes were incompletely corticated throughout (Figure 15C–E). The darker, central
axial filament could often be seen from the surface view of newer axes near branchlet tips
(Figure 15F). In cross-section, nodes had about 5 periaxial cells that were slightly larger than
the abundant, surrounding cortical cells (Figure 15G). Periaxial cells were generally small
enough so that it was possible to take a cross-section at a node that did not include the
periaxial cells and only revealed cortical cells (Figure 15H). The branchlet tips were often
slightly inrolled and pincer-shaped (Figure 15I–K); however, sometimes, the branchlet tips
were straight and not recurved at all (Figure 15L). These characteristics are consistent with
the original description of Ceramothamnion translucidum [
36
]. Furthermore, the relatively
smaller size of periaxial cells in Ceramothamnion translucidum separates it from species of
the genus Ceramium, whose periaxial cells are often more numerous and are generally
much larger and distinct from cortical cells at nodes [67].
Diversity 2024,16, 554 33 of 42
Diversity 2024, 16, x FOR PEER REVIEW 34 of 43
Figure 14. Ceramium plenatunicum collected from Fogland Beach, Tiverton, RI. (A,B) General habit
of thallus in situ. (C) Thallus in the lab. (D) Detail of wide-angled, spreading dichotomous branching
paern. (E,F) Axes are corticated throughout but may still appear banded, as cortical cells at nodes
appear as dark bands in surface view. (G) Branching is dichotomous throughout. (H) Cross-section
at node revealing seven circular periaxial cells (numbered). (I) Branchlet tips are not strongly re-
curved in these thalli, but they are still pincer-shaped.
Figure 15. Ceramothamnion translucidum collected from Ninigret Pond and Horseneck Beach. (A,B)
General habit of thallus. (C) Detail of the branching paern. (D,E) Branching is dichotomous and
mostly acute, often around 60° angles. (F) Detail of cortication at nodes, along with the clearly visible,
dark central axial filament (arrowheads). (G) Cross-section at a node showing five periaxial cells
(numbered) that are only slightly larger than the surrounding cortical cells. (H) Cross-section at a
node showing a single layer of cortical cells and no periaxial cells. (I–L) Variation in branchlet ter-
mini. Note the dark central axial filament visible in each branch.
Remarks: Based on molecular and morphological results, this species is a strong
match to Ceramothamnion translucidum, formally described in April 2024, from material
collected in New Brunswick [66]. Sequences generated in this study match those of speci-
mens collected in North Carolina (Figure 7A,B), as well as specimens from the Gulf of St.
Lawrence [66]. Considering the range of localities from which this species has been col-
lected and molecularly validated, both from our study and from others, as well as MARI-
Figure 15. Ceramothamnion translucidum collected from Ninigret Pond and Horseneck Beach.
(A,B) General habit of thallus. (C) Detail of the branching pattern. (D,E) Branching is dichoto-
mous and mostly acute, often around 60
◦
angles. (F) Detail of cortication at nodes, along with the
clearly visible, dark central axial filament (arrowheads). (G) Cross-section at a node showing five
periaxial cells (numbered) that are only slightly larger than the surrounding cortical cells. (H) Cross-
section at a node showing a single layer of cortical cells and no periaxial cells. (I–L) Variation in
branchlet termini. Note the dark central axial filament visible in each branch.
Remarks: Based on molecular and morphological results, this species is a strong match
to Ceramothamnion translucidum, formally described in April 2024, from material collected in
New Brunswick [
66
]. Sequences generated in this study match those of specimens collected
in North Carolina (Figure 7A,B), as well as specimens from the Gulf of St. Lawrence [
66
].
Considering the range of localities from which this species has been collected and molec-
ularly validated, both from our study and from others, as well as MARI-03342, which
was collected in 1972, this taxon is likely established in the NBA and has been present
for multiple decades. This species may have been historically overlooked or confused
with the similar C. deslongchampsii, which is reported to have 4 large periaxial cells, incom-
plete cortication, and forms mats on rocks and algae in the mid to low intertidal [
17
]. No
specimens that matched the morphology or matched existing published DNA barcodes
of C. deslongchampsii were collected in this study. However, it is still reported in the area
by numerous sources [
17
,
34
–
36
]. MARI-03342 had originally been identified as Ceramium
Diversity 2024,16, 554 34 of 42
fastigiatum, which is now a synonym of C. cimbricum, a species whose type locality is
Denmark and to which MARI-03342 and the rest of the Ceramothamnion specimens are
a poor molecular match [
16
]. C. cimbricum has been reported from Connecticut and the
NBA [
17
,
68
], but this study did not uncover any specimens that were a molecular match to
any European sequence data of this species.
Chondria littoralis Harvey 1853/Chondria sedifolia Harvey 1853
Molecular Results: Sequences were obtained from 13 specimens, and for 10 of these
specimens, both rbcL-3P and UPA sequences were generated. The rbcL-3P BLAST search
results were a 100% match to C. littoralis KF672853 from North Carolina (Figure 5A),
and the UPA BLAST search results were a 97.02% match to Chondria sp. MF101429
(Figure 5B). The intraspecies divergence for the rbcL-3P and UPA markers was 0–0.15%
and 0–0.27%, respectively (Table 6). The rbcL-3P and UPA markers disagreed on what the
nearest neighbor we collected was, the former being closest to C. baileyana and the latter
being closest to C. atropurpurea, with divergences from these species being 6.34–8.56% and
3.52–3.87%, respectively.
Locality and Morphology: This species was collected throughout the summer and
into the fall from as early as 23 June to as late as 12 October. Thalli were abundant on open
coast sites as lithophytes in subtidal waters, but occasionally young thalli were found as
epiphytes on Zostera (Table 7). Also often collected in the drift. Tetrasporophytes were
collected on 23 June 2022 and 1 August 2023; a single male gametophyte was collected on
29 July 2023, and female gametophytes were collected from mid-July to October. Thalli
were bushy with thick axes and visibly blunt branchlet tips, and they varied greatly in
color from straw yellow to a deep purplish maroon (Figure 16A–G). Axes were thick,
heavily corticated throughout and had 5 pericentral cells in cross-section (Figure 16H–L).
This species was easily distinguished from other Chondria in the NBA, as the apices of
branchlet tips were concave with the apical cells usually hidden in a sunken pit (Figure 16M).
Trichoblasts were absent in some thalli (Figure 16M), while in others, they were abundant,
highly dichotomously branched, and often had small, dark, pigmented cells interspersed
throughout the trichoblasts at branching dichotomies (Figure 16N). Tetrasporangia were
interspersed throughout the cortex of branchlet tips without much pattern (Figure 16O).
Cystocarps were usually found on distal growth on ultimate branchlets, but occasionally
on distal main axes. They were urn-shaped and were borne on pedestal-like lateral stalks,
upon which cystocarps developed adaxially (Figure 16P). Spermatangial sori were small,
hyaline plates that were organized in whorls around ultimate branchlet tips (Figure 16Q).
Remarks: There are many taxonomic issues with the genus Chondria in the NBA.
Four species have long been reported from the NBA, including C. baileyana,C. capillaris,
C. dasyphylla, and C. sedifolia. Three species were recovered in this study: C. baileyana,C.
atropurpurea, and C. littoralis/sedifolia. There were no issues with the identity and presence of
C. baileyana in the NBA, as the recovered specimens matched the morphological descriptions
of C. baileyana and were barcoded to C. baileyana with no disagreements (Figure 5A). Also,
in a recent paper, old herbarium specimens that had been initially identified and used
as the basis of the presence of C. capillaris in the NBA were determined to actually be C.
atropurpurea, a species previously thought to be confined to the Gulf of Mexico and the
Caribbean [
45
]. Similarly, this study found that all C. capillaris-like specimens from the
NBA had DNA barcodes that matched C. atropurpurea (Figure 5A). These three species—
C. baileyana,C. capillaris, and C. atropurpurea—comprise the exposed-apical-cell Chondria
group, and based on this study, only C. baileyana and C. atropurpurea are present in the
NBA (Figure 2). That leaves the sunken-apical-cell Chondria group, C. dasyphylla, C. sedifolia,
and C. littoralis/sedifolia. According to Mathieson & Dawes (2017), C. dasyphylla and C.
sedifolia can be differentiated by the thallus being “soft” or “tough,” by looking at the size
of the thallus, axis diameter, branchlet length, or the presence/absence of trichoblasts.
However, the numerous specimens we collected represented a gradient across all the size
and physical characteristics described by Mathieson & Dawes (2017), and they all barcoded
Diversity 2024,16, 554 35 of 42
to one species with very low sequence divergence. Considering this, the molecular and
morphological data suggest that there is only one species of morphologically plastic sunken-
apical-cell, Chondria, present in the NBA. Molecularly, this species matches C. littoralis
(rbcL-3P, Figure 5A) and Chondria sp. (UPA, Figure 5B). C. littoralis is a southern species
reported throughout the Caribbean and Gulf of Mexico [
16
], a situation reminiscent of C.
atropurpurea, as discussed above. On another note, there are public rbcL-3P sequence data
for C. dasyphylla available, and our sequences are at most a 94.94% and 97.99% match to that
species for rbcL-3P (MH388513, Figure 5A) and UPA (MT898439, Figure 5B), respectively.
Therefore, the sunken-apical-cell Chondria recovered in this study are not C. dasyphylla.
However, there are no published sequence data for C. sedifolia, so there is a chance that the
C. littoralis sequence to which our data matches could be a misidentified C. sedifolia. This is
supported by the morphological characterization of C. littoralis. C. littoralis is apparently
an exposed-apical-cell Chondria [
69
], which is inconsistent with the morphology of the
sunken-apical-cell species recovered in this study. In conclusion, the identity of this species
is still unclear; however, there is at least (likely at most) one species of sunken-apical-cell
Chondria that has been molecularly validated and has likely been present in the NBA for a
long time based on historical reports (Figure 2).
Diversity 2024, 16, x FOR PEER REVIEW 36 of 43
group, and based on this study, only C. baileyana and C. atropurpurea are present in the
NBA (Figure 2). That leaves the sunken-apical-cell Chondria group, C. dasyphylla, C. sedi-
folia, and C. lioralis/sedifolia. According to Mathieson & Dawes (2017), C. dasyphylla and C.
sedifolia can be differentiated by the thallus being “soft” or “tough,” by looking at the size
of the thallus, axis diameter, branchlet length, or the presence/absence of trichoblasts.
However, the numerous specimens we collected represented a gradient across all the size
and physical characteristics described by Mathieson & Dawes (2017), and they all bar-
coded to one species with very low sequence divergence. Considering this, the molecular
and morphological data suggest that there is only one species of morphologically plastic
sunken-apical-cell, Chondria, present in the NBA. Molecularly, this species matches C. lit-
toralis (rbcL-3P, Figure 5A) and Chondria sp. (UPA, Figure 5B). C. lioralis is a southern
species reported throughout the Caribbean and Gulf of Mexico [16], a situation reminis-
cent of C. atropurpurea, as discussed above. On another note, there are public rbcL-3P se-
quence data for C. dasyphylla available, and our sequences are at most a 94.94% and 97.99%
match to that species for rbcL-3P (MH388513, Figure 5A) and UPA (MT898439, Figure 5B),
respectively. Therefore, the sunken-apical-cell Chondria recovered in this study are not C.
dasyphylla. However, there are no published sequence data for C. sedifolia, so there is a
chance that the C. lioralis sequence to which our data matches could be a misidentified
C. sedifolia. This is supported by the morphological characterization of C. lioralis. C. lio-
ralis is apparently an exposed-apical-cell Chondria [69], which is inconsistent with the mor-
phology of the sunken-apical-cell species recovered in this study. In conclusion, the iden-
tity of this species is still unclear; however, there is at least (likely at most) one species of
sunken-apical-cell Chondria that has been molecularly validated and has likely been pre-
sent in the NBA for a long time based on historical reports (Figure 2).
Figure 16. Chondria lioralis/sedifolia collected from numerous open coast sites in the NBA. (A) Habit
of thallus in situ. (B) Detail of branchlet in situ. (C–E) Thalli in lab from Second Beach, Newport, RI,
Sheep Point Cove, Newport, RI, and Horseneck Beach, Westport, MA. (F) Detail of branchlets in lab.
(G) Prostrate branchlets. (H,I) Surface view of lower axes, which are heavily corticated. (J,K) Surface
view of cortical cells that are longitudinally elongated. (L) Cross-section showing five pericentral
Figure 16. Chondria littoralis/sedifolia collected from numerous open coast sites in the NBA. (A) Habit
of thallus in situ. (B) Detail of branchlet in situ. (C–E) Thalli in lab from Second Beach, Newport, RI,
Sheep Point Cove, Newport, RI, and Horseneck Beach, Westport, MA. (F) Detail of branchlets in lab.
(G) Prostrate branchlets. (H,I) Surface view of lower axes, which are heavily corticated. (J,K) Surface
view of cortical cells that are longitudinally elongated. (L) Cross-section showing five pericentral
cells (numbered) and a few layers of cortical cells. (M) Ultimate branchlets are blunt and have apical
pits that often obscure the apical cell. These branchlets had no trichoblasts. (N) Ultimate branchlets
with many trichoblasts, some of which have dark pigmentations in a few cells. (O) Tetrasporangia are
interspersed throughout the axes of ultimate branchlets or distal regions of main axes near the branch
termini. (P) Cystocarps are subspherical and are borne on small lateral pedestals. (Q) Spermatangial
sori are oriented around the apices of ultimate branchlets.
Diversity 2024,16, 554 36 of 42
Dasya pedicellata (C. Agardh) C. Agardh, 1824
Molecular Results: Sequences were generated for six specimens, and for three of these,
both rbcL-3P and UPA sequences were generated. The rbcL-3P sequences were a 100%
match to D. pedicellata ON002436 from Rhode Island (Figure 5A); UPA sequences were a
100% match to D. baillouviana HQ421392 from Rhode Island, with Dasya sp. HQ421299
being the second closest, which was a 98.10% match (Figure 5B). Considering that D.
baillouviana is an invalid name, the UPA GenBank voucher D. baillouviana HQ421392 may
well be D. pedicellata. Furthermore, since our rbcL-3P data matched D. pedicellata ON002436,
and the locality of that specimen was Rhode Island, the six specimens recovered in this
study are named D. pedicellata. The intraspecies divergence for both DNA markers was 0%,
and the species closest to D. pedicellata that we collected was D. elegans, which differed from
this species by 1.00–1.16% and 1.63% for the rbcL-3P and UPA markers, respectively.
Locality and Morphology: All specimens were collected from four open coast sites.
Thalli were abundant from late July to late October and were most frequently encountered
as a subtidal lithophyte. Some younger, developing thalli were found as epiphytes on
Zostera or coarse algae, but the largest individuals were always found growing on hard
substrata (Table 7). Many thalli were frequently found to be free-floating in the drift as well.
Tetrasporophytes and female gametophytes were collected throughout the late summer
from late July to October; however, male gametophytes were only collected in late October.
The thalli were most often a deep rose red and grew to be large, flowy bushes that were
easily recognizable due to their fuzzy appearance and bright coloration (Figure 17A,B).
Lateral branches were abundant, giving the thallus a much denser, fuller structure than that
of D. elegans (Figure 17C,D). The axes were heavily corticated and covered in uniseriate,
pigmented, dichotomously branched adventitious branchlets (Figure 17E–H). In cross-
section, the axes had 5–6 pericentral cells (Figure 17I). Developing cystocarps were often
narrow and cone-shaped, but fully developed cystocarps were large, urn-shaped with
clearly tapered, somewhat umbonate ostioles, clearly visible to the naked eye even when not
fully developed, and borne off the main corticated axes (Figure 17J,K). Both spermatangial
stichidia and tetrasporangial stichidia were abundant and borne from the adventitious
branchlets on male gametophytes and tetrasporophytes, respectively (Figure 17L–N). While
the morphologies of D. pedicellata and D. cf. elegans were very similar and may differ in the
ways discussed above under the D. cf. elegans treatment, the localities and seasonality of
these species seem to be different. D. cf. elegans was only collected from calm, estuarine
waters in the early summer, whereas D. pedicellata was only collected in the late summer at
open coast sites. However, this could simply be an artifact of having only collected Dasya
from warm, protected, estuarine waters in the early summer and only collecting Dasya
from open coast areas in the late summer. Both species may be present throughout the
entire summer and spring, and considering the sparsity of Dasya collected from estuarine
waters, D. pedicellata may also be present in these habitats. Further collections and work are
needed to fully flesh out the ecology of these two species.
Remarks: The rbcL-3P sequence data support the determination of D. pedicellata, whose
type locality is in New York and was first described two hundred years ago [
70
]. While D.
cf. elegans may be an introduced species, D. pedicellata is likely not and has been reported
from the area for many years under the invalid name D. baillouviana. For further discussion
on NBA Dasya spp., see remarks under D. cf. elegans (Section Dasya elegans (G. Martens) C.
Agardh Dasya elegans).
Diversity 2024,16, 554 37 of 42
Diversity 2024, 16, x FOR PEER REVIEW 38 of 43
Figure 17. Dasya pedicellata collected from various open coast or protected open coast NBA sites.
(A,B) General habit of thallus in situ at King’s Beach, Newport, RI, and Fort Wetherill, Jamestown,
RI. (C,D) Thalli in lab collected from Lighthouse Beach, Chatham, MA, and Sheep Point Cove, New-
port, RI. (E–G) Detail of axes covered in uniseriate adventitious branchlets. Dark cystocarps are
growing on the axes in (E,F,H) Detail of uniseriate adventitious branchlets. (I) Cross-section of the
main axis near the branch terminus revealing heavy cortication and six pericentral cells (numbered).
(J) Detail of axes with numerous immature cystocarps. (K) Mature cystocarp-releasing carpospores.
(L) Spermatangial stichidia are sessile and borne on uniseriate adventitious branchlets. (M) Detail
of axes with myriad tetrasporangial stichidia. (N) Tetrasporangial stichidia are borne on short stalks
on adventitious branchlets.
Remarks: The rbcL-3P sequence data support the determination of D. pedicellata,
whose type locality is in New York and was first described two hundred years ago [70].
While D. cf. elegans may be an introduced species, D. pedicellata is likely not and has been
reported from the area for many years under the invalid name D. baillouviana. For further
discussion on NBA Dasya spp., see remarks under D. cf. elegans (Section 3.4.1.4 Dasya ele-
gans).
4. Discussion
DNA barcoding has proved to be a useful tool for assessing the biodiversity of the
order Ceramiales in the NBA. Molecular results not only helped identify species inde-
pendent of overlapping morphologies, but they also provided insight into numerous
Figure 17. Dasya pedicellata collected from various open coast or protected open coast NBA sites.
(A,B) General habit of thallus in situ at King’s Beach, Newport, RI, and Fort Wetherill, Jamestown,
RI. (C,D) Thalli in lab collected from Lighthouse Beach, Chatham, MA, and Sheep Point Cove,
Newport, RI. (E–G) Detail of axes covered in uniseriate adventitious branchlets. Dark cystocarps are
growing on the axes in (E,F,H) Detail of uniseriate adventitious branchlets. (I) Cross-section of the
main axis near the branch terminus revealing heavy cortication and six pericentral cells (numbered).
(J) Detail of axes with numerous immature cystocarps. (K) Mature cystocarp-releasing carpospores.
(L) Spermatangial stichidia are sessile and borne on uniseriate adventitious branchlets. (M) Detail of
axes with myriad tetrasporangial stichidia. (N) Tetrasporangial stichidia are borne on short stalks on
adventitious branchlets.
4. Discussion
DNA barcoding has proved to be a useful tool for assessing the biodiversity of the order
Ceramiales in the NBA. Molecular results not only helped identify species independent
of overlapping morphologies, but they also provided insight into numerous taxonomic
discrepancies. Most discrepancies were a result of algae from the NBA being mistakenly
identified as morphologically similar but distinct species from abroad, mostly Europe. A
few examples of other local NBA Ceramiales not discussed in detail in this study that were
mistaken for extralimital species whose identities were resolved within the last few years
include Spyridia americana, which had long been misidentified in the NBA as the eastern
Diversity 2024,16, 554 38 of 42
Atlantic S. filamentosa, and Pleonosporium novae-angliae, which had been mistaken for P.
borreri [
24
,
44
]. This study also agreed with these works and provided new sequence data
that can be used to resolve outstanding taxonomic issues in the genera Ceramium and Dasya.
Furthermore, five species were reported from the NBA for the first time, including at least
two potentially undescribed species and at least two new reports of the introduced species.
The first potentially undescribed species, Streblocladieae sp., MARI-04466, loosely
matches the historical morphological records of Bryocladia subtilissima, but MARI-04466 was
molecularly distinct from this species (as reported in Genbank) and was not a close match
to any other published sequence data. The second unknown species, Antithamnion sp., for
which a couple of specimens were collected and sequenced, matched no historical morpho-
logical records or any published sequence data. The two new records of introduced species
in the NBA include Acanthosiphonia echinata and Antithamnionella spirographidis. A single
specimen, MARI-04540, was collected from Horseneck Beach, Westport, MA, and was both
a strong morphological and molecular match to A. echinata. This species was described from
Florida, but has only recently been reported in the Northwestern Atlantic (New Brunswick)
and has been reportedly introduced to the Mediterranean and
Indonesia [49,50,52]
. This
species may have been present in the NBA for a long time, but there is also a chance that
this species only recently migrated northward from the type locality as a result of global
warming or through other introduction vectors. Another singular specimen, MARI-04622,
was collected at Lighthouse Beach, Chatham, MA, and was a strong morphological match
and molecular match to A. spirographidis [
54
]. Unlike A. echinata, this species is likely native
to the Pacific and was introduced throughout
Europe [55,57]
. This is the first report of A.
spirographidis from the western Atlantic, indicating that this may be a newly introduced
species to the region.
While most of the sampling was conducted over the summer months, with some
sparse collecting in the fall of 2023, the majority of historically reported species from the
area were collected and molecularly validated (Figure 2). Further collections in the winter
and spring months, as well as more attempts at sequencing older herbarium specimens,
should be able to fill the sampling gaps and help determine if some of the historically
reported species not encountered in this study are no longer present.
Although there are still some gaps left to fill with respect to understanding ceramialean
biodiversity and ecology in the NBA, many new sequences as well as specimen-tied se-
quences were generated, which will serve as useful comparisons for future work involving
species of Ceramiales in the NBA and more distant waters. Furthermore, the UPA gene
has again been shown to be a reliable marker for separating species. The UPA marker
determined the same species identities as the traditionally used rbcL-3P marker, and there
was a universal barcoding gap where maximum intraspecies divergences were never above
the threshold of about 0.3% sequence divergence, which was below the minimum recorded
interspecies divergence of about 0.5% (but which was over 1.0% in every case, except for
Ceramium secundatum/virgatum). We conclude that there is a discrete limit for separating
ceramialean species with the UPA marker that is comparable to other more widely-used
markers like the rbcL-3P marker. This, along with the ease of amplification and sequencing
(Table 4), and its short length, makes the UPA a useful marker for specimen-independent
methods of biodiversity assessment and discovery.
Finally, the library of ceramialean UPA sequences generated here, which represent com-
mon fouling organisms with a propensity for survival beyond their native
ranges [15,33]
,
establishes an important foundation for detecting species in nature from nondescript dis-
persal stages (i.e., spores, gametes, zygotes, fragments). As this foundation continues to
grow, DNA metabarcoding using UPA sequences will become a strategic tool for rapid
biodiversity assessment to help understand shifts in biodiversity and the discovery of
non-native introduction pathways. In particular, the detection of marine species beyond
their native ranges is often ascribed to ballast water [
3
,
71
–
81
], and though this vector is
confirmed for many organismal groups, relatively few studies [
3
,
79
–
81
] have verifiably
established this for marine macroalgae. We think that DNA metabarcoding with the UPA
Diversity 2024,16, 554 39 of 42
marker holds great promise both for ballast water monitoring and for comprehensive as-
sessments of natural waters, but it necessarily relies on a library of DNA barcodes grounded
in vouchered, specimen-by-specimen assessment.
Author Contributions: B.W. conceived and designed the study, secured funding, generated data,
oversaw field collections, data management, and analysis, and undertook technical and editorial
review of the manuscript. T.I. drafted the original manuscript. T.I. and A.B. conducted field collections,
curated specimens, and generated and analyzed morphological and DNA sequence data. All authors
have read and agreed to the published version of the manuscript.
Funding: This project is a component of the Rhode Island Seaweed Biodiversity Project, funding for
which was provided by the Rhode Island Science and Technology Advisory Council and RI EPSCoR
(NSF Award #1004057). The latter awarded a Summer Undergraduate Research Fellowship to A.B.
Additional funding was provided by the Roger Williams University (RWU) Foundation to Promote
Scholarship & Teaching and an RWU Sabbatical award to B.W. Additional funding from RWU was
provided to T.I. in the form of a Senior Research Thesis supplies grant, and the Mark Gould Memorial
Summer Student Fellowship.
Institutional Review Board Statement: Not applicable.
Data Availability Statement: DNA sequence data are available in GenBank under the accession
numbers listed Table 5. Herbarium specimens are housed at Roger Williams University.
Acknowledgments: Students in the Fall 2015 Marine Phycology (BIO 355) and Genetics (BIO 200)
courses at RWU, as well as fellow undergraduate researchers, including Benjamin Carolan, Abigail St.
Jean, and Matthew Koch, are gratefully acknowledged for contributing specimens and/or data to
this project. We gratefully acknowledge the scholarly and editorial review of earlier versions of this
manuscript by Larry Liddle, Marcie Marston, and Koty Sharp.
Conflicts of Interest: The authors declare no conflicts of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results.
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