A Demonstration of DNA Barcoding-Based Identification of Blade-Form Ulva (Ulvophyceae, Chlorophyta) Species from Three Site in the San Juan Islands, Washington, USA

Abstract

Marine macroalgae are foundation species that play a critical ecological role in coastal communities as primary producers. The macroalgal genus Ulva is vital in intertidal communities, serving as a food source and shelter for organisms, but these species also form environment-damaging nuisance blooms. This project aimed to demonstrate the utility of DNA barcoding for determining the diversity of Ulva species in the San Juan Islands (Washington, DC, USA). Blade-form Ulva (Ulvophyceae) specimens were collected from the lower, mid, and upper intertidal zones at three sites experiencing different levels of wave exposure. Sequences of plastid-encoded tufA were generated for each specimen and cluster analyses revealed the presence of four species at the collection sites. Two species were positively identified as Ulva expansa and Ulva fenestrata based on their sharing identical tufA sequences with those of the holotype specimens. Sequences of plastid-encoded rbcL and the nuclear-encoded ribosomal ITS regions of representative specimens were used to identify the other two species as Ulva prolifera and Ulva californica based on their similarity to epitype and topotype specimen sequences, respectively. Additional types of specimen sequencing efforts are needed to increase the number of Ulva species that can be accurately identified and realize their true biodiversity.

Full text

Citation: Kuba, G.M.; Carpio-
Aguilar, B.; Eklund, J.; Freshwater,
D.W. A Demonstration of DNA
Barcoding-Based Identification of
Blade-Form Ulva (Ulvophyceae,
Chlorophyta) Species from Three Site
in the San Juan Islands, Washington,
USA. Diversity 2022,14, 899. https://
doi.org/10.3390/d14110899
Academic Editors: Manuel
Elias-Gutierrez and Jun Sun
Received: 25 July 2022
Accepted: 20 October 2022
Published: 24 October 2022
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diversity
Article
A Demonstration of DNA Barcoding-Based Identification of
Blade-Form Ulva (Ulvophyceae, Chlorophyta) Species from
Three Site in the San Juan Islands, Washington, USA
Gabrielle M. Kuba 1,2,3, Brenda Carpio-Aguilar 1,4, Jason Eklund 1,5 and D. Wilson Freshwater 1,6 ,*
1Friday Harbor Marine Laboratory, University of Washington, Friday Harbor, WA 98250, USA
2Department of Biology, College of Charleston, Charleston, SC 29424, USA
3Department of Biological and Environmental Sciences, University of Rhode Island, Kingston, RI 02881, USA
4School of Biology, Universidad de Costa Rica, San Jose 10102, Costa Rica
5Department of Botany, Connecticut College, New London, CT 06320, USA
6Center for Marine Science, University of North Carolina at Wilmington, Wilmington, NC 28403, USA
*Correspondence: freshwaterw@uncw.edu; Tel.: +1-910-962-2375
Abstract:
Marine macroalgae are foundation species that play a critical ecological role in coastal
communities as primary producers. The macroalgal genus Ulva is vital in intertidal communities,
serving as a food source and shelter for organisms, but these species also form environment-damaging
nuisance blooms. This project aimed to demonstrate the utility of DNA barcoding for determining
the diversity of Ulva species in the San Juan Islands (Washington, DC, USA). Blade-form Ulva
(Ulvophyceae) specimens were collected from the lower, mid, and upper intertidal zones at three sites
experiencing different levels of wave exposure. Sequences of plastid-encoded tufA were generated
for each specimen and cluster analyses revealed the presence of four species at the collection sites.
Two species were positively identified as Ulva expansa and Ulva fenestrata based on their sharing
identical tufA sequences with those of the holotype specimens. Sequences of plastid-encoded rbcL
and the nuclear-encoded ribosomal ITS regions of representative specimens were used to identify
the other two species as Ulva prolifera and Ulva californica based on their similarity to epitype and
topotype specimen sequences, respectively. Additional types of specimen sequencing efforts are
needed to increase the number of Ulva species that can be accurately identified and realize their
true biodiversity.
Keywords: ITS; macroalgae; rbcL; tufA;Ulva californica;Ulva expansa;Ulva fenestrata;Ulva prolifera
1. Introduction
The northeast Pacific Ocean, from the coasts of Southeast Alaska to Oregon, is charac-
terized by a diverse community of marine algae, including 671 taxa and 284 genera [
1
]. The
San Juan Islands within the Salish Sea are a particularly rich area within this region that
experience mixed semidiurnal tides that cause intense tidal flows with vigorous vertical
mixing, especially at sills [
2
,
3
]. The characteristics of channels through the islands are
highly influenced by the Fraser River from the Strait of Georgia [
4
], and the succession
of spring and neap tides modulates the mixing over the sills, regulating the estuarine
exchange of water [
3
]. The mixture of cold ocean waters of high salinity with brackish
surface waters, seasonality, and physical factors further supports the diversity of the marine
community and affect the interaction among resident organisms [
5
]. This is especially true
for marine macroalgae, which have highly diverse intertidal and subtidal communities
in this region. The diversity of these organisms can be masked by the high frequency of
cryptic and phenotypically plastic species [6,7].
Marine macroalgae are foundation species that play a critical ecological role in coastal
communities as primary producers and habitat-defining organisms [
8
]. Ulva Linnaeus
Diversity 2022,14, 899. https://doi.org/10.3390/d14110899 https://www.mdpi.com/journal/diversity

Diversity 2022,14, 899 2 of 11
species are important components of biodiversity and bioindicators [
9
] However, they
have also been associated with the majority of blooms of free-floating green algae responsi-
ble for ‘green tides’ because Ulva species can rapidly grow in nutrient-rich habitats and
have a high tolerance range for abiotic factors such as temperature and salinity [
6
,
10
–
13
].
Eutrophication-driven green tides in shallow waters have a direct economic impact on
coastal communities, making it essential to identify the species involved for bloom charac-
terization and control [
10
,
14
]. In addition, it is important to understand their potential uses
in pharmaceutical applications for drug development [
12
], as well as in biotechnological
and industrial processes as bioremediators, biofuels, and food sources [
14
]. However,
their simple morphology and phenotypic plasticity means that diversity assessments and
identifications of Ulva species based on morphological characters range from challenging
to impossible e.g., [15,16].
The genus Ulva is constituted of nearly 100 taxonomically accepted species [
17
] includ-
ing those species previously placed in Enteromorpha Link [
18
]. This green algal genus is
present in both freshwater and marine environments. In the latter, it is ubiquitous along
coasts, rocky shores, and protected bays and estuaries, growing attached to substrata
or found as drift. The morphological characterization of Ulva species has traditionally
included both macro- and microscopic features. Macroscopic features include having distro-
matic blade-form or monostromatic tubular thalli, thallus shape, size, extent of branching
and presence or absence of marginal dentation. Cellular features considered key to identifi-
cation include cellular shape and dimensions, number of pyrenoids, arrangement of the
cells in regular or irregular patterns, and thallus thickness e.g., [
19
–
22
]. Although previous
studies used these characters for identification, they have been found to vary within species
depending on thallus age, reproductive state, wave exposure, tidal factors, temperature,
salinity, light, life-history stage, and biological factors such as herbivory and associated
microbiome e.g., [
9
,
23
,
24
]. In addition, the morphological plasticity of Ulva species results
in a variety of forms and ecotypes. Therefore, the taxonomic status of species in this genus
remains uncertain and difficult to assess [9,11,16,24,25].
Molecular analysis of Ulva spp. is greatly expanding our understanding of their tax-
onomic and phylogenetic status [
25
]. Studies utilizing DNA sequence data have defined
many molecular-based species e.g., [
11
,
16
,
26
,
27
], but sequences from type specimens have
been generated for relatively few historical species [
25
,
28
–
30
] and only recently have type
sequences been included in new species descriptions [
16
,
31
,
32
]. The historical types that
have been sequenced demonstrate that very few of the specimen identifications for se-
quences in public databases are correct [
30
,
33
]. Accordingly, while Ulva species can be easily
delimited with DNA sequence data, the identification of most species remains problematic.
Up to 17 species and varieties of Ulva (including taxa formerly classified as Enteromor-
pha) have been reported in the northeast Pacific [
34
]. Hayden and Waaland [
6
] reported
12 species based on molecular and morphological analyses in the most recent treatment of
the genus from this region. Little is known about Ulva species in the San Juan Islands; how-
ever, multiple studies have focused on the surrounding Salish Sea ecosystem [
6
,
35
–
38
]. Ulva
species within this area proliferate into blooms comprised of multiple species in the inter-
tidal zone, similar to many other anthropogenically influenced coastal ecosystems
[39–42]
.
They were found to outcompete other macroalgae within these zones, exhibiting harmful
characteristics that alter species interactions [
42
,
43
]. A better understanding of the species
involved is needed for these reasons.
DNA barcoding was originally envisioned as a utilitarian method that could be simply
applied for the identification of species by a non-specialist using a single universal marker,
the mitochondria-encoded cytochrome c oxidase subunit 1 gene 5
0
region [
44
,
45
]. While
this vision has been realized for many groups of organisms, others have been found to
require different and multiple markers [
46
,
47
]. Ulva and other green algae are part of
the latter group, but studies have shown that plastid-encoded rbcL and tufA, as well as
nuclear-encoded ITS, are useful singly or in combination for barcoding these algae
[48–50]
.
The objectives of this study were to demonstrate the utility of DNA barcoding in its

Diversity 2022,14, 899 3 of 11
simplest application to determine the number and, if possible, identity of Ulva species.
This was achieved by exploring the diversity of blade-form Ulva species present at three
environmentally different study sites in the San Juan Islands, Washington.
2. Materials and Methods
2.1. Sample Collection
Thirty-five blade-form Ulva specimens were collected from the intertidal zone at three
sites of differing relative wave exposure within the San Juan Islands, Washington (Table 1).
Collections were made within the low, mid, and high intertidal zones at each location. Spec-
imens were chosen at each location based on observed macromorphological variation, and
two algal specimens of representative morphologies identified in each intertidal zone were
collected at each sampled site. Specimens were only collected if attached and not as drift,
and transported on ice back to the lab, where they were placed into a running seawater table
until processed. Each specimen was morphologically identified using the Gabrielson and
Lindstrom [
1
] key and vouchers were made and deposited in the University of Washington
herbarium (WTU). All specimen data, including photographic images, are available from
the Barcode of Life Database system website (dx.doi.org/10.5883/DS-MASJI08).
Table 1. Ulva specimen collection site information.
Site Name Wave Exposure Level Latitude, Longitude Date
Iceberg Point, Lopez Is. High 48.42◦N, 122.90◦W 25 June 2021
Cattle Point, San Juan Is. Mid 48.45◦N, 122.96◦W 27 June 2021
Friday Harbor Lab, San Juan Is. Low 48.55◦N, 123.01◦W 29 June 2021
2.2. DNA Extraction, Amplification, and Sequencing
Total DNA was extracted from specimens using a Bioline Extract-PCR Kit (Bioline,
Taunton, MA, USA) following the protocol of Taylor et al. [
50
], with small modifications as
follows. Approximately 0.5 cm
2
of healthy blade tissue was chopped into small pieces, and
incubated at 75
◦
C in 50
µ
L of Extract-PCR kit enzymatic solution for 1–20 h before enzyme
deactivation by heating at 95
◦
C for 10 min. Cellular debris was pelleted by centrifugation
and samples were diluted 1:10 and stored at −20 ◦C.
The plastid-encoded tufA locus was amplified for each Ulva specimen using MyTaq
HS Red Mix following the manufacturer’s protocol (Bioline) with primers described in
Fama et al. [
51
]. Cycling conditions were as follows: an initial denaturing step of 95
◦
C for
2:45 min, followed by 35 cycles of 95
◦
C for 15 s, 45
◦
C for 15 s, and 72
◦
C for 1 min, with a
final extension at 72
◦
C for an additional 4 min. PCR products were enzymatically cleaned
using Exo-Sap (Thermo Fisher Scientific, Waltham, MA, USA) and sent to Genewiz for DNA
sequencing (Azenta Life Sciences, South Plainfield, NJ, USA). Based on initial analyses of
tufA sequences, nuclear-encoded ITS and plastid-encoded rbcL sequences were generated
from representative specimens of the detected species. ITS and rbcL were amplified and
sequenced following the protocols of Freshwater et al. [
52
] but using a MyTaq HS Red DNA
Polymerase Kit (Bioline), and the ITS and rbcL primers described by Shimada et al. [
53
].
Individual sequence reactions were compiled and edited using Sequencher (v. 5.4, Gene
Codes Corporation, Ann Arbor, MI, USA).
2.3. DNA Sequence Analyses and Species Identifications
Alignments of DNA sequences were generated using MUSCLE [
54
] as implemented
in MEGA (v. 7.0.26, [
55
]) or Geneious (v. 9, Biomatters Limited, Aukland, New Zealand).
Species were molecularly delineated through barcode sequence clustering. Initially a
UPGMA cluster diagram was generated from an alignment of the 35 tufA sequences for
the newly collected San Juan Islands Ulva specimens to establish specimen clusters. Inter-
and intra-cluster sequence divergence values were then assessed to determine if there were
barcode gaps, as defined by Meier et al. [
56
] between clusters and whether these barcode

Diversity 2022,14, 899 4 of 11
gaps fit the tufA species divergence threshold ranges of Saunders and Kucera [
49
] and
Kirkendale et al. [
57
]. GenBank BLAST analyses [
58
] of the tufA and, where needed, ITS
and rbcL sequences were used to explore the identifications of the resulting molecularly
defined species.
3. Results
The 35 Ulva specimens were grouped into four species based on UPGMA cluster
analysis of tufA sequences (Figure 1). Intraspecific variation in tufA sequences was only
seen in Species-1 (0.0–0.7%; 3 haplotypes) and interspecific variation among the four species
ranged from 3.2–3.3% to 7.5% (Table 2). BLAST searches revealed that tufA sequences of
Species-3 and Species-4 were exact matches to those of the U. expansa (Setchell) Setchell
& N.L. Gardner (GenBank # MH731007) and U. fenestrata Postels & Ruprecht (GenBank #
MK456404) type specimens, respectively. BLAST searches of the Species-1 and Species-2
tufA sequences returned close matches to specimens within the U. linza-procera-prolifera
(LPP) complex clade for Species-1 and specimens predominantly identified as U. californica
Wille for Species-2.
Diversity 2022, 14, x FOR PEER REVIEW 5 of 11
Figure 1. UPGMA tufA cluster analysis for 35 blade-form Ulva specimens collected at different in-
tertidal zones from three sites in the San Juan Islands. Specimen labels include the collection number
(e.g., ‘03UA’) followed by the morphological identification of the specimen based on the Gabrielson
and Lindstrom [1] key. The 1% sequence divergence level is indicated by the grey vertical line and
example images of specimens are shown on the right.
Table 2. Intra- and interspecific divergences among tufA sequences from 35 specimens of four blade-
form Ulva species collected in the San Juan Islands, WA. Gray background = intraspecific diver-
gences; white background = interspecific divergences.
Species-1
Species-2
Species-3
Species-4
U. prolifera
U. californica
U. expansa
U. fenestrata
n = 12
n = 6
n = 9
n = 8
Species-1
0.0–0.7%
U. prolifera
n = 12
Species-2
3.2–3.3% 0.00%
U. californica
n = 6
Species-3
6.2–6.4% 6.2–6.3% 0.00%
U. expansa
n = 9
Species-4
6.9–7.2% 7.50% 5.4–5.5% 0.00%
U. fenestrata
n = 8
Figure 1.
UPGMA tufA cluster analysis for 35 blade-form Ulva specimens collected at different
intertidal zones from three sites in the San Juan Islands. Specimen labels include the collection
number (e.g., ‘03UA’) followed by the morphological identification of the specimen based on the
Gabrielson and Lindstrom [
1
] key. The 1% sequence divergence level is indicated by the grey vertical
line and example images of specimens are shown on the right.

Diversity 2022,14, 899 5 of 11
Table 2.
Intra- and interspecific divergences among tufA sequences from 35 specimens of four blade-
form Ulva species collected in the San Juan Islands, WA. Gray background = intraspecific divergences;
white background = interspecific divergences.
Species-1 Species-2 Species-3 Species-4
U. prolifera U. californica U. expansa U. fenestrata
n= 12 n= 6 n= 9 n= 8
Species-1
U. prolifera
n= 12
0.0–0.7%
Species-2
3.2–3.3%
U. californica
n= 6
0.00%
Species-3
6.2–6.4% 6.2–6.3%
U. expansa
n= 9
0.00%
Species-4
6.9–7.2% 7.50% 5.4–5.5%
U. fenestrata
n= 8
0.00%
The ITS sequences of Species-1 specimens representative of tufA haplotype 1 (specimen
03UA) and haplotype 2 (specimen 20UA) were identical. There was only a single base-
pair difference between the ITS-2 region of this sequence and the ITS-2 sequences of
17 U. prolifera O.F. Müller topotype specimens (GenBank# AJ012276, but see discussion),
including the epitype designated by Cui et al. [
59
]. The ITS-2 region sequence of the
Species-1 specimen representative of tufA haplotype 3 (specimen 14UB) is two base pairs
different from that of the U. prolifera epitype.
The rbcL sequences of two representative specimens of Species-2 (09UB; 23UB) were
identical to each other and a topotype specimen identified by Hayden et al. [
18
] as U. cali-
fornica (GenBank #AY255866). Similarly, ITS sequences of specimens 09UB and 23UB were
identical and only 0.7% different from the ITS sequence of the Hayden et al. [
18
] topotype
specimen identified as U. californica (GenBank #AY260560).
4. Discussion
Distinguishing Ulva species is a well-known problem in phycology. They have a
very simple morphology and the few morphological characters that have been used to
describe species exhibit intraspecific variation e.g., [
15
,
16
,
21
,
60
,
61
]. Analyses of DNA
sequences are currently popular for delineating Ulva species e.g., [
9
,
62
,
63
]. However, as
clearly demonstrated in a series of papers by Hughey et al. [
25
,
29
,
30
], Ulva specimens
can only confidently be identified to species if DNA sequence data from those specimens
can be matched to that of type specimens. These papers, as well as the overall analysis
of Ulva sequences in GenBank by Fort et al. [
33
], demonstrated that many of the names
assigned to Ulva sequences in public databases are incorrect. As an extreme example, all
sequences in GenBank assigned to U. rigida were incorrectly identified [
30
]. Fort et al. [
33
]
identified accessions that could be used for species identifications when a query sequence
was homologous, and Hughey et al. [
30
] provide a table with all sequenced historical types
and sequence determined synonyms.
Analyses of tufA sequences for blade-form Ulva specimens from different tidal heights
at three different locations in the San Juan Islands revealed the presence of four species
(
Figure 2
). Two of these species could be positively identified through the homology of their
tufA sequences to that from type specimens. One was identified based on the holotype se-
quences published by Hughey et al. [
28
] as U. expansa (type locality:
Monterey, CA, USA
), a
species reported in an older floristic survey of nearby Whidbey Island [
35
] and by Scagel [
34
]
in his flora of British Columbia and northern Washington. Although reported as far north as
British Columbia in these and other treatments of Northeast Pacific macroalgae e.g., [
64
–
66
],
Tanner [
36
] synonymized U. expansa with U. fenestrata based on the morphological variation
observed in herbarium specimens, field collections and culture studies. This synonymy

Diversity 2022,14, 899 6 of 11
was followed in the recent keys to the marine algae from southeastern Alaska to Oregon
that have functioned as de facto floras for this region in recent years [
1
,
67
,
68
]. However,
Hughey et al. [
25
,
28
] demonstrated the distinction of these two species. and U. expansa was
once again recognized in the Northeast Pacific flora e.g., [69].
Diversity 2022, 14, x FOR PEER REVIEW 6 of 11
4. Discussion
Distinguishing Ulva species is a well-known problem in phycology. They have a very
simple morphology and the few morphological characters that have been used to describe
species exhibit intraspecific variation e.g., [15,16,21,60,61]. Analyses of DNA sequences
are currently popular for delineating Ulva species e.g., [9,62,63]. However, as clearly
demonstrated in a series of papers by Hughey et al. [25,29,30], Ulva specimens can only
confidently be identified to species if DNA sequence data from those specimens can be
matched to that of type specimens. These papers, as well as the overall analysis of Ulva
sequences in GenBank by Fort et al. [33], demonstrated that many of the names assigned
to Ulva sequences in public databases are incorrect. As an extreme example, all sequences
in GenBank assigned to U. rigida were incorrectly identified [30]. Fort et al. [33] identified
accessions that could be used for species identifications when a query sequence was ho-
mologous, and Hughey et al. [30] provide a table with all sequenced historical types and
sequence determined synonyms.
Analyses of tufA sequences for blade-form Ulva specimens from different tidal
heights at three different locations in the San Juan Islands revealed the presence of four
species (Figure 2). Two of these species could be positively identified through the homol-
ogy of their tufA sequences to that from type specimens. One was identified based on the
holotype sequences published by Hughey et al. [28] as U. expansa (type locality: Monterey,
California, USA), a species reported in an older floristic survey of nearby Whidbey Island
[35] and by Scagel [34] in his flora of British Columbia and northern Washington. Alt-
hough reported as far north as British Columbia in these and other treatments of North-
east Pacific macroalgae e.g., [64–66], Tanner [36] synonymized U. expansa with U. fenestrata
based on the morphological variation observed in herbarium specimens, field collections
and culture studies. This synonymy was followed in the recent keys to the marine algae
from southeastern Alaska to Oregon that have functioned as de facto floras for this region
in recent years [1,67,68]. However, Hughey et al. [25,28] demonstrated the distinction of
these two species. and U. expansa was once again recognized in the Northeast Pacific flora
e.g., [69].
Figure 2. Map showing the distribution of blade-form Ulva species collected from different intertidal
heights at three San Juan Island sites that experience different wave exposures. Species are indicated
Figure 2.
Map showing the distribution of blade-form Ulva species collected from different intertidal
heights at three San Juan Island sites that experience different wave exposures. Species are indicated
by color and intertidal zones by letters: L = low; M = middle; H = high. Locations: FHL = Friday
harbor Laboratory beach front; CP = Cattle Point; IP = Iceberg Point.
The second positively identified species was determined to be U. fenestrata (type
locality: Kamchatka, Russia) based on the tufA sequence for its holotype published by
Hughey et al. [
25
]. Similar to U. expansa,U. fenestrata was included in the Scagel [
34
]
flora and Tanner’s [
36
] treatment of Northeast Pacific Ulva, but not in the more recent
comprehensive keys [
1
,
67
,
68
]. Ulva fenestrata has generally been identified based upon the
presence of perforations in the blade e.g., [
65
,
70
], but whether the presence or absence of
perforations is a true developmental characteristic of species has been questioned, and
both perforated and non-perforated specimens have been included in this species [
36
,
70
].
Gabrielson et al. [
68
] included U. fenestrata-type perforated blades within an unidentified
Ulva species given the place-holder name U. “lactuca”. Further unpublished observations
of San Juan Islands Ulva specimens led to this species being considered to represent
U. fenestrata [71], and the current study verifies this identification.
The remaining two species revealed by the tufA analysis did not have close homology
to any currently available tufA sequences from an Ulva type or topotype specimen. The tufA
sequences from specimens of one of these species included three different haplotypes that
had close homology to GenBank sequences from specimens placed in the Ulva linza-procera-
prolifera (LPP) complex clade, a group composed of specimens variously identified as
U. linza Linnaeus, U. procera (K.Ahlner) H.S.Hayden, Blomster, Maggs, P.C.Silva, Stanhope
& Waaland, and U. prolifera O.F.Müller. Cui et al. [
59
] used morphological, molecular and
crossing studies to examine the status of LPP complex specimens collected from Lolland
Island, Denmark, the type locality of U. prolifera. Combining their results with those of
previous LPP-complex-related studies e.g., [
62
,
72
,
73
], it was determined that U. prolifera was
best represented by tubular branched specimens with sexual or asexual life histories [
59
].
The lectotype of U. prolifera is a drawing in Müller [
74
], thus an epitype was designated
and sequences for the ITS-2 and 5S rDNA spacer regions generated. The ITS-2 sequence

Diversity 2022,14, 899 7 of 11
of this specimen was not made publicly available, but was reported to be identical to a
previously published sequence with GenBank accession number AJ012276. The AJ012276
sequence includes not only ITS-2 but also the ITS-1 and 5.8S rRNA regions and, therefore,
the epitype ITS-2 sequence is only the 215 bp portion of the AJ012276 sequence between
the annealing sites of the two primers used by Cui et al. [
59
] to amplify and sequence this
region in their specimens. ITS-2 sequences for representative specimens of the LPP complex
species collected in the San Juan Islands were only 1–2 base pairs different (0.47–0.93%)
from that of the U. prolifera epitype. This divergence from the epitype ITS-2 sequence is less
than or equal to that of any LPP complex specimens included in the Cui et al. [59] study.
The San Juan Islands specimens were identified as U. prolifera based on these results.
However, an unquestioned identification will require an understanding of whether any
taxa within the LPP complex clade represent U. linza. Interestingly, the San Juan Islands
specimens molecularly identified in this study as U. prolifera, were not branched tubes, the
characteristic morphology of the species, but distromatic blades that became tubular where
they were basally narrow near the point of attachment. This latter morphology has been
identified as U. linza in the Northeast Pacific e.g., [
1
,
64
,
68
], and all these specimens were
morphologically identified as U. linza (Figure 1). Similar to U. prolifera the lectotype of
U. linza is an illustration [
75
] (pl. 9, Figure 6), but there is an epitype in OXF. Unfortunately,
requests for the minimal type specimen material needed for current DNA sequence genera-
tion techniques have not been fulfilled, and the status of U. linza remains unresolved [
76
].
Regardless, the findings herein indicate that the concept of U. prolifera needs to be expanded
to include blade-form thalli.
Representative specimens of the fourth species resolved in this study by the tufA
analysis had rbcL sequences that were identical to that generated by Hayden et al. [
18
]
from a La Jolla, California specimen of U. californica, the type locality for this species. The
ITS sequences of San Juan Island specimens and that of this topotype specimen were also
closely homologous and varied by only four base pairs (0.7%). The Hayden et al. [
18
]
topotype specimen (WTU 344798) agrees with the type specimen (US 57108) in being ca.
2 cm or less and having turfy blades, and provides a basis for molecularly identifying
specimens as U. californica in lieu of sequence data from the type specimen.
Tanner [
60
] conducted field, herbarium and culture studies of U. californica,U. angusta
Setchell & N.L. Gardner, and U. scagelii Chihara, three morphologically similar Northeast
Pacific species that differed in size, habit and distribution. The results of these studies led to
the synonymy of U. angusta and U. scagelii with U. californica, increasing the size range and
geographic distribution of U. californica. Specimens of U. californica sequenced in this study
were also variable in size and distribution (Figure 2; dx.doi.org/10.5883/DS-MASJI08).
The close homology of the topotype DNA sequences with those of specimens from this
and other studies, e.g., [
6
,
49
,
77
], verifies the wider Northeast Pacific distribution and the
environmentally determined morphological variation found in the study of Tanner [60].
As demonstrated in this study, simple analyses of DNA barcode sequences can be
a useful tool for quickly distinguishing Ulva species, and only with an understanding of
the number and extent of these species can applied questions concerning their diversity,
ecology and physiology be addressed. For example, determining the species composition
of blooms, or establishing monocultures or mixed cultures for industrial applications.
However, this in no way diminishes the importance of extensive specimen collection
combined with thorough phylogenetic and species delimitation analyses, e.g., [
16
,
26
], to
establish the species and sequence characteristics upon which utilitarian DNA barcoding
methods are based. The application of names to barcode-defined species, however, remains
problematic. Two of the four species included in this study could be positively identified
because DNA sequences were publicly available for their holotype specimens, and the best
current identifications were possible for the other two species based on DNA sequences of
epitype and topotype specimens. This fortuitous result is unusual because so few historical
Ulva types have been sequenced, and only additional type specimen sequencing efforts and

Diversity 2022,14, 899 8 of 11
cooperation of the herbaria housing Ulva types can ensure that the application of additional
species names is accurate.
Author Contributions:
Conceptualization, G.M.K., B.C.-A. and D.W.F.; methodology, G.M.K.,
B.C.-A.
and D.W.F.; validation, G.M.K., B.C.-A., J.E. and D.W.F.; formal analysis, G.M.K., B.C.-A., J.E. and
D.W.F.; investigation, G.M.K., B.C.-A., J.E. and D.W.F.; resources, D.W.F.; data curation, G.M.K.,
B.C.-A.
, J.E. and D.W.F.; writing—original draft preparation, G.M.K., B.C.-A. and J.E.;
writing—review
and editing, G.M.K., B.C.-A. and D.W.F.; visualization, G.M.K. and D.W.F.; supervision, D.W.F.; project
administration, G.M.K., B.C.-A., J.E. and D.W.F.; funding acquisition, D.W.F. All authors have read
and agreed to the published version of the manuscript.
Funding:
Research conducted at the University of Washington’s Friday Harbor Marine Laboratory
was funded by the Marine Botany class budget and research conducted the University of North
Carolina at Wilmington’s Center for Marine Science was supported by the CMS DNA-Algal Trust.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement:
The data presented in this study are openly available in the BOLD
system database at dx.doi.org/10.5883/DS-MASJI08 and GenBank accessions OP347101-OP347108;
OP347119-OP347153; OP347156-OP347160.
Acknowledgments:
We would like to thank Tom Mumford for his guidance and mentorship during
the Friday Harbor Laboratory Marine Botany class. We would also like to thank the Friday Harbor
Laboratory for their facilities, resources, and accessibility during this study. Multiple reviewers
provided valuable guidance for this publication.
Conflicts of Interest: The authors declare no conflict of interest.
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