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Article

DNA Barcode-Assisted Inventory of the Marine Macroalgae from the Azores, Including New Records

by
Daniela Gabriel
1,2,*,
William E. Schmidt
3,
Joana Micael
4,
Mónica Moura
1 and
Suzanne Fredericq
3
1
Faculty of Sciences and Technology, University of the Azores, 9500-321 Ponta Delgada, Portugal
2
BIOPOLIS Program in Genomics, Biodiversity and Land Planning, CIBIO, 4485-661 Vairão, Portugal
3
Department of Biology, University of Louisiana at Lafayette, Lafayette, LA 70504-3602, USA
4
Southwest Iceland Nature Research Centre, 245 Suðurnesjabær, Iceland
*
Author to whom correspondence should be addressed.
Phycology 2024, 4(1), 65-86; https://0-doi-org.brum.beds.ac.uk/10.3390/phycology4010004
Submission received: 22 December 2023 / Revised: 13 January 2024 / Accepted: 17 January 2024 / Published: 19 January 2024
Browse Figures
Versions Notes

Abstract

:
Up to the present study, only 8.5% of the 522 macroalgal species reported at the Azores have sequences deposited in GenBank and BOLD public repositories. The sequences of four genetic markers (cox1, rbcL, UPA, tufA) were obtained for recently collected samples from two Azorean islands. DNA barcode-assisted identification was conducted on newly generated and unpublished sequences from public repositories. A literature review of recently published studies, including the molecular identifications of Azorean macroalgae, was also performed. The results confirm the occurrence of 51 species (including subspecific ranks) and provide four new records, namely, three cryptogenic species (Olokunia boudouresquei, Padina gymnospora, and Ulva lacinulata) and one introduced species (Ulva australis). This study contributes 23 DNA barcodes generated for the first time to the Azores, which now has 10.5% of its marine flora represented in public repositories. Additionally, UPA sequences were generated for the first time for the five taxa.

1. Introduction

The Azores Archipelago, located in the mid-Atlantic ridge, is composed of nine islands separated into three main groups (eastern, central, and western), spread over 620 km from SE to NW, 1465 km from mainland Portugal and 1925 km from Newfoundland (Canada; Figure 1A). The marine flora of the Azores has been extensively studied since 1843 [1]. Currently, it comprises 522 macroalgal taxa [2], of which 97 belong to the Ochrophyta, 349 to the Rhodophyta, and 76 to the Chlorophyta, with the number of species known to occur on each island varying from 43 in Corvo to 328 in São Miguel [3,4]. Regarding the origin of the taxa, 42 are currently reported as introduced species [2], from which 9 are classified as invasive [5], 43 as cryptogenic or of unknown origin [3,6,7], and the remaining 437 being possibly native, from which 8 are Azorean endemic [7].
Macroalgae is a term used to refer to macroscopic, aquatic, photosynthetic organisms composed of filaments or simple tissues belonging to three different Phyla: brown algae (Ochrophyta), red algae (Rhodophyta), and green algae (Chlorophyta; [8]). The identification of macroalgal species is usually challenging due to the simple morphology and anatomy, the high rates of convergence, high phenotypic plasticity, and the limited information on life histories [9]. Comparative DNA sequences analyses have been used to assist the morphology-based studies of macroalgae since the 1990s [10,11,12], and the support of molecular markers became the standard approach to assess the algal species-level diversity [13]. Several studies involving the molecular-assisted identification have contributed to revealing the molecular diversity of the macroalgae occurring in the Azores (as listed in Section 3.3).
DNA barcoding is an identification system based on the comparison between the sequence of a genetic marker from a given specimen against a database of sequences from an a priori defined species [13]. The variety of markers used in DNA-assisted morphological studies presents a challenge in comparing newly sequenced specimens with the sequences available in public databases [9]. A few standard markers are currently used as barcodes for macroalgae, mainly rbcL for the three Phyla, combined with cox1 for Ochrophyta and Rhodophyta, and tufA for Chlorophyta [14]. Sequencing from different species and worldwide populations, as well as the provision of the links between sequences and proper species names [13], are needed to build an extensive DNA barcode library to facilitate the easy identification of algae for ecological, legislative, and commercial purposes [14].
The present study aims to provide an inventory of the marine macroalgae with species identity supported by molecular data reported from the Azores. The inventory is based on sequences deposited in GenBank and BOLD public repositories, as well as newly sequenced vouchers. The native statuses regarding the species’ origins were evaluated for the Azores, and the year of the first reports to these islands were provided. This study also intends to increase the sequences available for DNA barcoding from the Azores Archipelago in public databases.

2. Materials and Methods

2.1. Sampling

Samples were collected by hand at low tide from five sites in São Miguel and three sites in Santa Maria Islands (Western Group; Figure 1 and Figure S1; Table 1 and Table S1) between June 2020 and September 2022. Specimens were photographed in situ (underwater) and/or in vivo (in a container with seawater or wet on herbarium paper) using an Olympus Tough TG-5 waterproof camera (OM Digital Solutions Corporation, Tokyo, Japan; Figure 2 and Figure S2). In each case, a voucher was pressed onto herbarium paper, with subsamples kept in silica gel for subsequent molecular work. Vouchers are stored with Daniela Gabriel (address above), who performed a preliminary identification based on general morphology and will eventually be deposited in the Ruy Telles Palhinha Herbarium at the University of the Azores, Ponta Delgada (Code AZB).

2.2. DNA Extraction, PCR Amplification, and Sequencing

Total DNA was extracted using the methods described by [15]. PCR amplification was performed in 20 μL reaction mixtures with MyTaq Polymerase and Mix (Bioline) following the manufacturer’s instructions: 4 μL of MyTaq Mix (enhanced buffer containing dNTPs and MgCl2), 1 μL of MyTaq Polymerase, 1 μL of each primer (10 μM), 2 μL of DNA, and 11 μL of H2O. Published primers were used to amplify the mitochondrion-encoded gene Cytochrome c oxidase subunit 1 DNA barcode region (cox1, ~664 bp; [9]) and the chloroplast-encoded genes ribulose-1,5-bisphosphate carboxylase large subunit (rbcL, ~1350 bp; [16,17,18,19]), elongation factor tu (tufA, ~800 bp; [20,21]), and LSU (23S) domain V (UPA, ~370 bp; [22]—Table S2). PCR conditions were optimized and are as follows: (1) rbcL: initial denaturation at 95 °C for 3 min, followed by 35 cycles of 94 °C for 45 s, 47 °C for 45 s, and 72 °C for 90 s, with a final extension of 72 °C for 10 min; (2) tufA: initial denaturation at 94 °C for 3 min, followed by 40 cycles of 94 °C for 20 s, 52 °C for 15 s, and 72 °C for 55 s, with a final extension of 72 °C for 5 min; (3) UPA: initial denaturation at 95 °C for 2 min, followed by 35 cycles of 95 °C for 20 s, 55 °C for 30 s, and 72 °C for 30 s, with a final extension of 72 °C for 10 min; and (4) cox1: initial denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 1 min, 45 °C for 1 min, and 72 °C for 1 min, with a final extension of 72 °C for 10 min. Sanger DNA sequencing was performed with StabVida (Lisbon, Portugal) using the produced PCR fragments with the respective sets of primers.
Forward and reverse sequence reads were edited and concatenated using Geneious Prime 2023.2 (Biomatters Inc., Boston, MA, USA) and later deposited in GenBank (https://0-www-ncbi-nlm-nih-gov.brum.beds.ac.uk/genbank; accessed on 21 December 2023), with the accession numbers OR944632-OR944652 and OR961484-OR961498. Newly generated sequences were compared to those on the GenBank sequence database through Basic Local Alignment Search Tool—NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi; accessed on 21 December 2023). Sequences with more than 99% identity match were considered to belong to the same species [23]. Sequence names from GenBank were confirmed with the most recent publications containing these sequences and the most updated taxonomy available on Algaebase (https://www.algaebase.org; accessed on 21 December 2023).

2.3. Compilation of Publicly Available Sequences for the Azores

The information of DNA sequences currently available on GenBank and Barcode Of Life Data System—BOLD (https://www.boldsystems.org; accessed on 21 December 2023), hereafter public repositories, with “Azores” included in the sampling locality, were retrieved. Records retrieved from BOLD linked to GenBank suppressed sequences were not considered. Unpublished sequences, i.e., sequences deposited in public repositories but not included in the peer-reviewed literature, were submitted to BLAST for identification. The sequences of vouchers identified at the species level were used to produce an inventory of the Azorean marine flora supported by molecular identification. Sequence names were updated as described in Section 2.2 and classified according to their native distributional range as described in Section 2.4.

2.4. Evaluation of Native Status in the Azores

Taxa confirmed to be present in the Azores were classified as native, i.e., occurring within the species’ native distributional ranges; endemic, i.e., occurring only in the Azores; introduced, i.e., occurring outside the species native distributional range; invasive, i.e., with documented impacts on the Azorean biodiversity; or cryptogenic, i.e., not clearly classified as native or introduced in the archipelago. The species known distributional ranges were retrieved from Algaebase, and their eventual report as being invasive or posing a threat of invasion elsewhere was checked in the databases Information system on aquatic non-indigenous and cryptogenic species—AquaNIS (http://www.corpi.ku.lt/databases/index.php/aquanis; accessed on 21 December 2023), SeaLifeBase (www.sealifebase.org), Centre for Agriculture and Bioscience International Invasive Species Compendium—CABI: (www.cabi.org/ISC; accessed on 21 December 2023), Global Invasive Species Database—GISD (www.iucngisd.org; accessed on 21 December 2023), European Alien Species Information Network—EASIN (https://easin.jrc.ec.europa.eu/easin; accessed on 21 December 2023), and Invasive and Exotic Species of North America list—IESNA (www.invasive.org; accessed on 21 December 2023). The date of the first record of each species in the Azores is also provided.

3. Results

3.1. DNA-Barcoding of Newly Generated Sequences

A total of 36 sequences from four different markers were produced for the 24 collected samples, namely, ten sequences of cox1, four of tufA, seven of rbcL, and 15 of UPA (Table S1). The newly generated sequences comprise 24 barcodes for 14 species present in the Azores, with 22 originally published barcodes (Appendix A). UPA barcodes belonging to five species are published here for the first time (Laurencia pyramidalis, Jania pedunculata var. adhaerens, Jania verrucosa, Gelidium microdon, and Lomentaria articulata).

3.2. Analyses of Publicly Available Sequences

A total of 373 DNA sequences from Azorean vouchers were retrieved from public repositories, namely, 147 sequences of cox1, ten of cox2, 74 of cox3, 14 of LSU, two of nad1, four of psaA, 17 of psbA, 89 of rbcL, eight of rbcL-rbcS spacer, one of rpl16, and seven of tufA (Table S3). Unpublished sequences were subject to molecular analyses to assess their correct identities (Appendix B). Of the 58 taxa retrieved, 13 were identified only at the generic level. The assembled publicly available sequences comprise 66 barcodes for 45 species present in the Azores.

3.3. DNA-Based Species Inventory

The molecular analyses of the newly generated sequences (Section 3.1) and the sequences retrieved from public repositories (Section 3.2) uncovered 55 species (including subspecific ranks), with 20 species belonging to Ochrophyta, 28 to Rhodophyta, and 7 to Chlorophyta (Table 2). The uncovered taxa represent 51 known species of Azorean marine flora and 4 new species: 3 cryptogenic species (Olokunia boudouresquei, Padina gymnospora, and Ulva lacinulata) and 1 introduced species (Ulva australis).

3.4. Updates to the Azorean Marine Flora

The most recent update to the number of marine macroalgae in the Azores was published by Gabriel et al. [2], who reported the occurrence of 522 taxa (97 Ochrophyta, 349 Rhodophyta, and 76 Chlorophyta). The revision of the recent peer-reviewed literature revealed that three species should be added (Pseudoralfsia azorica, Dictyota pleiacantha, and Cystoseira pustulata) and two species should be removed (Cystoseira compressa and C. humilis) from that account based on new species descriptions, new reports, or taxonomic clarification of those taxa [25,36,40]. Together with the new reports of the present study (Olokunia boudouresquei, Padina gymnospora, Ulva australis, and Ulva lacinulata), the updated marine flora comprises 527 species (99 belonging to Ochrophyta, 350 to Rhodophyta, and 78 to Chlorophyta).

4. Discussion

The present study increases our knowledge of the Azorean marine flora and increases the number of taxa with sequences deposited in public repositories from 45 to 55. The temporal trend of sequences published in GenBank indicates a substantial increase since 2021 (Figure 3). Because the submission date of sequences deposited in BOLD is not specified, we were unable to detect such a trend in this public repository. The number of marine macroalgae known to the Azorean was also raised from 522 to 527. The number of introduced species reported to the Azores has also increased from 42 to 43, while the number of cryptogenic species has increased from 43 to 46.
Only 10.5% of the Azorean marine flora is currently represented in public repositories, which poses a limitation for barcoding studies, including environmental barcoding (e-barcoding). This percentage may be underestimated since some authors fail to inform the voucher locality when submitting sequences to public repositories. The examples of sequences from the Azores that were not retrieved in our search (as described in Section 2.3) are those from Corallina berteroi, Cystoseira pustulata, Vertebrata barbarae, and V. cymatophila, and species were added to the inventory only because the authors knew the publications where they were published [35,49,50]. The identification of taxa deposited in public repositories is to be considered with caution since accuracy cannot always be verified in the articles where the sequences were published, and the sequences of type material are seldom available [85,86].
Most sequences from Azorean vouchers retrieved from public repositories were published in taxonomic studies and produced in the context of phylogenetic reappraisals (e.g., [25,28,36,45,47,48,49,56,59,61,83]. Besides the fact that morphological observations supported the identifications, these studies sometimes provided the sequences of type material (e.g., Dictyota pleiacantha and Laurencia pyramidalis). For the newly generated sequences, the standard barcodes cox1, rbcL, and tufA were enough to discriminate the species [14], and the sequences of type material were used whenever available (e.g., Hypnea musciformis and Ulva lacinulata).
UPA, a universal marker for photosynthetic organisms, was also used in this study despite the low efficacy of this genetic marker to discriminate certain closely related species [14,87]. Easily amplified with a single pair of primers, UPA can identify a general group of algae and indicate the choices of primers to sequence other informative markers [88]. For that reason, UPA sequences are provided for most newly generated sequences, with five species receiving their first sequence of this marker. We were unable to produce DNA sequences other than UPA for only two vouchers, namely, Padina gymnospora and Sytosiphon lomentaria. Therefore, a further sequencing of other genetic markers is necessary to confirm the species’ identities.
The groups of macroalgae have different percentages of taxa represented in public repositories when compared to the known marine flora of the Azores. Ochrophyta has 20% of its taxa with known sequences, having the highest followed by Chlorophyta with 9% and Rhodophyta with 8% (Figure 4). This variation reflects the fact that most studies that generated the molecular data (Table 2) focused on specific taxa. This is the first study to provide DNA barcodes for Azorean macroalgae from vouchers that were not specifically targeted but were collected for being conspicuous in the sampling sites.
Sampling was performed in the initial stages of the Rugulopteryx okamurae invasion on the southern coast (2020) of São Miguel Island [4,30]. As the invasion progressed, sampling was performed in the initial stages of the Rugulopteryx okamurae invasion on the northern and western coasts (2022; [4,30]). No specimen of Rugulopteryx okamurae was observed during the samplings performed on Santa Maria Island, though the collectors were not focused on searching for this species. The sampled taxa are not easily observed now due to the mentioned invasion.
Even though this was a small DNA-barcoding study, i.e., based on the collection of conspicuous specimens from a small number of sites, only in the intertidal zone of two islands, this study contributed to increasing the percentage of taxa from the Azorean marine flora represented in public repositories from 8.5% to 10.5%. Besides contributing to DNA barcodes from the Azores, the study also led to the uncovering of four new records for the archipelago, showing that the Azorean marine flora is still poorly known.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/phycology4010004/s1, Figure S1: Collection sites. São Miguel: (A) Caloura; (B) Atalhada; (C) Praia do Pópulo; (D) São Vicente de Ferreira; and (E) Mosteiros. Santa Maria: (F) Maia; (G) Praia Formosa; and H) Anjos; Figure S2: In vivo pictures of some marine macroalgae from the Azores analyzed in the present study. Ochrophyta: (A) Colpomenia sinuosa; (B) Fucus spiralis; (C) Padina gymnospora; and (D) Scytosiphon lomentaria. Rhodophyta: (E) Amphiroa beauvoisii; (F) Gelidium microdon; (G) Hypnea musciformis; (H) Laurencia pyramidalis; and (I) Lomentaria articulata. Chlorophyta: (J) Ulva australis; (K) Ulva compressa; and (L) Ulva lacinulata. Scale bar = 1 cm; Table S1: Collection information and GenBank accession numbers for the newly collected samples used; Table S2: Primers and PCR conditions for the different genetic markers used [9,16,17,18,19,20,21,22]; Table S3: Sequences from Azorean vouchers retrieved from GenBank and BOLD, with the respective references [3,4,24,25,28,31,36,40,43,45,47,48,50,56,58,59,60,61,64,66,70,81,89,90].

Author Contributions

Conceptualization, D.G. and J.M.; methodology, D.G., J.M. and M.M.; formal analysis, D.G. and J.M.; investigation, D.G., J.M. and W.E.S.; data curation, D.G.; writing—original draft preparation, D.G., J.M. and S.F.; writing—review and editing, D.G., J.M., W.E.S., M.M. and S.F.; funding acquisition, D.G. and M.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the AÇORES 2020 Operational Program (85% by the European Regional Development Fund and 15% by the Autonomous Region of the Azores Budget) under Grant ACORES-01-0145-FEDER-000091. DG was supported by the Foundation for Science and Technology research Grant DL 57/2016/CP1440/CT0025. CIBIO-Açores is maintained with Portuguese National Funds through the Foundation for Science and Technology under Project UIDB/50027/2020.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available in Supplementary Tables S1 and S3. DNA sequences can be retrieved from GenBank and BOLD using the accession numbers in the mentioned tables.

Acknowledgments

The present work was generated in the context of Project CRYPTO—Molecular Identification of Cryptogenic Macroalgae with Invasive Potential in the Azores. The authors would like to thank António Medeiros for providing the map, Ana Isabel Ferreira, Beatriz Martins, and Cláudia Ribeiro for helping with DNA sequencing, and Ana Filipa Santos, Ana Isabel Ferreira, and Susana Cabral for helping with collections. We also thank the two anonymous reviewers for their constructive suggestions.

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.

Appendix A

DNA barcode-assisted analysis of samples collected and sequenced in this study as described in Section 3.1 (Table A1).
Table A1. DNA barcode-assisted analysis of samples collected and sequenced in this study. Sequences marked with * represent the first DNA sequence of the marker for the species based on Azorean vouchers, and sequences marked with + represent the first DNA sequence of the marker for the species.
Table A1. DNA barcode-assisted analysis of samples collected and sequenced in this study. Sequences marked with * represent the first DNA sequence of the marker for the species based on Azorean vouchers, and sequences marked with + represent the first DNA sequence of the marker for the species.
TaxaGenetic MarkerGenBank
Accession
Numbers		Top MatchesComments
Ochrophyta
Colpomenia sinuosarbcL *OR944643
OR944644		Colpomenia sinuosa, 99.39% similarity, Hawaiian Islands (AB578988) and Korea (AY398468)
UPA *OR961485Colpomenia sinuosa, 99.74% similarity, China (OR094252) and Hawaiian Islands (EF426596)
Fucus spiraliscox1OR944632Fucus spiralis, 100% similarity, the Atlantic USA coast (EU646722)
Fucus vesiculosus, 100% similarity, Norway (MN184303)		Phylogeny reconstructions based on complete mitochondrion and chloroplast genomes support the recognition of Fucus spiralis var. vesiculosus [91], although this taxon is not currently recognized in Algaebase. Until further taxonomic decisions, and since the presence of F. vesiculosus in the Azores is not confirmed by morphological studies [7], F. spiralis is maintained as the identity of the Azorean voucher.
UPA *OR961486
OR961487
OR961488		Fucus spiralis, 99.75% similarity, the Pacific USA coast (MG922855)
Fucus vesiculosus, 99.75% similarity, Portugal (FM957154)
Padina gymnosporaUPA *OR961489Padina gymnospora, 99.20% similarity, Hawaiian Islands (EF426588)UPA sequences from Padina pavonica, the only Padina species currently reported to the Azores, are not available for comparison.
Scytosiphon lomentariaUPA *OR961490Scytosiphon promiscuous, 100% similarity, Japan (MK107984)
Hapterophycus canaliculatus, 100% similarity, Korea (MF591718)
Petalonia binghamiae (as Endarachne binghamiae), 100% similarity, Korea (MF460360)
Scytosiphon lomentaria, 100% similarity, China (MK798154)
Petalonia fascia, 100% similarity, the Atlantic USA coast (KF367780) and China (KC782886)		Phylogeny reconstructions based on a complete chloroplast genome show that Scytosiphon promiscuus and Petalonia binghamiae are different taxa [92], though UPA sequences retrieved from their complete chloroplast genome are 100% similar.
Rhodophyta
Amphiroa beauvoisiicox1 *OR944633
OR944634
OR944635
OR944636		Amphiroa beauvoisii, 100% similarity, Brazil (MG521348)Phylogenetic reconstructions based on four genetic markers indicate that Amphiroa beauvoisii is a species complex [87], with the Azorean vouchers nested in “clade 2”.
UPA *OR961491
OR961492		Amphiroa beauvoisii, 100% similarity, Brazil (MG521307) and the Atlantic USA coast (KF367784)
Gelidium microdonrbcL *OR944645Gelidium microdon, 100% similarity, the Atlantic Spanish coast (KT920271)
UPA *+OR961493This is the first UPA sequence for Gelidium microdon
Hypnea musciformiscox1 *OR944637
OR944638		Hypnea musciformis, 99.85% similarity, Tunisia (MG030775) and Italy (MF597663)A sequence from the complete chloroplast genome generated from a topotype of Hypnea musciformis was used for comparison (OL964154; [93]).
rbcL *OR944646Hypnea musciformis, 99.93% similarity, Italy (OL964154), France (EU346011), and the Mediterranean Spanish coast (KT428787)
UPA *OR961494Hypnea musciformis, 100% similarity, Italy (OL964154) and the Atlantic USA coast (KJ202096)
Jania pedunculata var. adhaerenscox1 *OR944639Jania sp., 100% similarity, the Indian South African coast (LC071780)
Jania adhaerens, 99.85% similarity, China (OP168510)		Based on the lectotype, Jania pedunculata var. adhaerens should be referred to as a variety rather than a distinct species [52].
UPA *+OR961495This is the first UPA sequence for Jania pedunculata var. adhaerens
Jania verrucosacox1 *OR944640Jania verrucosa, 98.95% similarity, the Indian South African coast (LC071781)Although the similarity between cox1 sequences was slightly beyond 99%, this match was used to discriminate the species due to the higher similarity observed between the rbcL sequences.
rbcL *OR944647Jania verrucosa, 99.89% similarity, New Zealand (KM369140)
UPA *+OR961496This is the first UPA sequence for Jania verrucosa
Laurencia pyramidalisrbcLOR944648Laurencia pyramidalis, 100% similarity, the Atlantic France (FJ785316), the Canary Islands (KF492791), the Azores (KF492787), and the Mediterranean Turkish coast (MZ153331)
UPA *+OR961497This is the first UPA sequence for Laurencia pyramidalis
Lomentaria articulatacox1 *OR944641Lomentaria articulata, 100% similarity, the Atlantic French coast (KJ960811)
UPA *+OR961498This is the first UPA sequence for Lomentaria articulata
Chlorophyta
Ulva australistufA *OR944649Ulva australis, 100% similarity, the Atlantic French coast (MT160640), Italy (LR732070), the Netherlands (MZ272366), and Australia (MK125443)
Ulva pertusa, 100% similarity, China (MN853875)
Ulva compressatufA *OR944650Ulva compressa, 99.85% similarity, Australia (JN029285)
Ulva lacinulatatufA *OR944651
OR944652		Ulva lacinulata, 100% similarity, Croatia (MW543061) and Portugal (MT160657)
U. laetevirens, 100% similarity, Italy (MF544109), Tunisia (MF614791), and Korea (MT625105)		A sequence from the complete chloroplast genome generated from the lectotype of Ulva lacinulata was used for comparison (MW543061; [83]).

Appendix B

DNA barcode-assisted analysis of sequences deposited in public repositories but not included in the peer-reviewed literature as described in Section 3.2 (Table A2).
Table A2. DNA barcode-assisted analysis of sequences deposited in public repositories but not included in the peer-reviewed literature (unpublished sequences). Sequence marked with * represents the first DNA sequence of the marker for the species based on Azorean vouchers.
Table A2. DNA barcode-assisted analysis of sequences deposited in public repositories but not included in the peer-reviewed literature (unpublished sequences). Sequence marked with * represents the first DNA sequence of the marker for the species based on Azorean vouchers.
TaxaGenetic MarkerGenBank/
BOLD
Accession
Numbers		Top MatchesComments
Ochrophyta
Colpomenia sinuosacox1PHAZO004-10
PHAZO117-10
PHAZO118-10
PHAZO119-10
PHAZO203-10
PHAZO204-10		Colpomenia sinuosa, 99.6–100% similarity, Australia (MZ054832)
Cystoseira pustulatacox1OL434655
(as Cystoseira pustulata)
PHAZO043-10
(as Cystoseira compressa)		Cystoseira pustulata, 100% similarity, the Canary Islands (OK480304), the Azores (MF768044), and the Mediterranean Spanish coast (MF768042)
Fucus spiraliscox1PHAZO060-10
PHAZO062-10
PHAZO063-10		Fucus spiralis, 100% similarity, the Atlantic USA coast (EU646722)
Fucus vesiculosus, 100% similarity, Norway (MN184303)		Phylogeny reconstructions based on complete mitochondrion and chloroplast genomes support the recognition of Fucus spiralis var. vesiculosus [91], although this taxon is not currently recognized in Algaebase. Until further taxonomic decisions, and since the presence of F. vesiculosus in the Azores is not confirmed by morphological studies [7], F. spiralis is maintained as the identity of the Azorean voucher.
Gongolaria abies-marinacox1PHAZO156-10
(as Cystoseira abies-marina)		Gongolaria abies-marina, 100% similarity, Azores (OK480429)
Halopteris filicinacox1PHAZO003-10
PHAZO005-10
PHAZO008-10
PHAZO041-10
PHAZO042-10
PHAZO153-10		Halopteris filicina, 99.8% similarity, Mediterranean French coast (EU579868)
Halopteris scopariaLSUOL434652Halopteris scoparia, 99.2% similarity, Tunisia (OK646025)
Petalonia binghamiaecox1PHAZO002-10
(as Petalonia cf. fascia)		Petalonia binghamiae, 100% similarity, China (MF374731; as Endarachne binghamiae).
Sargassum cymosumcox1OL434657
PHAZO044-10
PHAZO045-10
PHAZO116-10
(as Sargassum sp.)		Sargassum natans, 100% similarity, Curaçao (OM460646), from the Northwestern Atlantic Ocean and the Caribbean Sea (KY084907—KY084908, KY084912
Sargassum fluitans, 100% similarity, from the Gulf of Mexico (MT470904)		Phylogenetic reconstructions based on cox1, ITS-2, and rbcL [94] indicate that both species should be regarded as synonyms of S. cymosum. This name is used despite the lack of a formal amendment to S. cymosum since the species is reported to the Azores [7], unlike S. natans and S. fluitans.
Scytosiphon lomentariacox1PHAZO060-10
PHAZO062-10
PHAZO063-10		Scytosiphon lomentaria, 99.8–100% similarity, Argentina (ON568311)
Zonaria tournefortiicox1OL434658Zonaria tournefortii, 100% similarity, the Canary Island (MW224392) and the Atlantic USA coast (KF367749)
Rhodophyta
Asparagopsis armatacox1OL434653Asparagopsis armata, 100% similarity, the Atlantic French coast (KJ960344) and the UK (AB774223)
Asparagopsis taxiformiscox1OL434654Asparagopsis taxiformis, 100% similarity, Italy (KJ398158) and the Mediterranean Spanish coast (AB774217)
Corallina berteroicox1 *HQ919507Corallina caespitosa, 100% similarity, the Atlantic Spanish coast (KF460944)Reappraisal of Corallina species based on type material [95] suggested that the cosmopolitan species C. caespitosa should be referred to as C. berteroi.
HQ919508Corallina caespitosa, 100% similarity, the Atlantic Spanish coast (KF460946) and the Atlantic French coast (KJ960596)
HQ919502
HQ919503
HQ919504
HQ919505
HQ919506
HQ919509
HQ919510
HQ919511		Corallina caespitosa, 99.2–99.6% similarity, the Azores (KP834357)
Olokunia boudouresqueirbcLEU349143
(as Cruoriopsis crucialis)		Olokunia boudouresquei, 100% similarity, New Zealand (KC998949; as Peyssonnelia boudouresquei)This sequence is reported as included in Krayesky et al. [60], but no reference to the sequence is found in this article.
Osmundea pinnatifidarbcLKU647694/
REDEU882-13		Osmundea pinnatifida, 100% similarity, Ireland (AF281875)
Pterocladiella capillaceacox1OL434656Pterocladiella capillacea, 100% similarity, Tunisia (MG030796) and the Mediterranean French coast (OL809719)
Chlorophyta
Caulerpa webbiana f. distichatufAFM956073Caulerpa webbiana, 100% similarity, Red Sea (AJ417958), Australia (KF314152), and the Hawaiian Islands (KJ957133)Further studies of the different formae within the species are needed to confirm the subspecific rank of the Azorean voucher.

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Phycology 04 00004 g001
Figure 1. Map of the study area. (A) Azores and its position in the North Atlantic; (B) The Azores Archipelago; (C) Santa Maria Island; and (D) São Miguel Island. Collection sites are marked with yellow circles (see Table 1 for further details).
Figure 1. Map of the study area. (A) Azores and its position in the North Atlantic; (B) The Azores Archipelago; (C) Santa Maria Island; and (D) São Miguel Island. Collection sites are marked with yellow circles (see Table 1 for further details).
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Phycology 04 00004 g002
Figure 2. In situ pictures of some marine macroalgae from the Azores analyzed in the present study. Ochrophyta: (A) Fucus spiralis; (B) Padina gymnospora; and (C) Scytosiphon lomentaria. Rhodophyta: (D) Amphiroa beauvoisii; (E) Jania verrucosa; and (F) Lomentaria articulata. Chlorophyta: (G) Ulva australis; (H) Ulva compressa; and (I) Ulva lacinulata.
Figure 2. In situ pictures of some marine macroalgae from the Azores analyzed in the present study. Ochrophyta: (A) Fucus spiralis; (B) Padina gymnospora; and (C) Scytosiphon lomentaria. Rhodophyta: (D) Amphiroa beauvoisii; (E) Jania verrucosa; and (F) Lomentaria articulata. Chlorophyta: (G) Ulva australis; (H) Ulva compressa; and (I) Ulva lacinulata.
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Figure 3. Cumulative number of taxa from Azorean vouchers with sequences deposited in GenBank. The number above line represents the sum of Ochrophyta, Rhodophyta, and Chlorophyta.
Figure 3. Cumulative number of taxa from Azorean vouchers with sequences deposited in GenBank. The number above line represents the sum of Ochrophyta, Rhodophyta, and Chlorophyta.
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Figure 4. Percentage of taxa known to the Azorean marine flora with sequences deposited in public repositories. Dark colors indicate taxa with sequences, and light colors indicate taxa without sequences.
Figure 4. Percentage of taxa known to the Azorean marine flora with sequences deposited in public repositories. Dark colors indicate taxa with sequences, and light colors indicate taxa without sequences.
Phycology 04 00004 g004
Table 1. Collection sites in the Azores.
Table 1. Collection sites in the Azores.
LocalityCollectorsDateCoordinates
São Miguel Island
Caloura, CercoD. Gabriel, A. F. Santos21, 24-July-202037.708154 N 25.509782 W
Atalhada, CruzeiroD. Gabriel9-June-202037.744306 N 25.594341 W
Livramento, Praia do PópuloD. Gabriel8-June-202037.750304 N 25.618740 W
São Vicente de Ferreira, harbourD. Gabriel27-September-202237.834432 N 25.668619 W
Mosteiros, tide poolsD. Gabriel28-September-202237.899311 N 25.821360 W
Santa Maria Island
MaiaD. Gabriel, A. I. Ferreira27-August-202236.939619 N 25.014179 W
Praia FormosaD. Gabriel, A. I. Ferreira25-August-202236.949958 N 25.094974 W
Anjos, Poça do CarroD. Gabriel, A. I. Ferreira, S. Cabral25-August-202237.005955 N 25.161788 W
Table 2. Inventory of the Azorean marine flora supported by molecular identification, with targeted genetic markers deposited in public repositories, first time reported, and the origin status in the Azores. Sequences deposited in public repositories but not included in peer-reviewed literature are referred to as “unpublished”. Taxa marked with “*” represent updates for the Azorean marine flora.
Table 2. Inventory of the Azorean marine flora supported by molecular identification, with targeted genetic markers deposited in public repositories, first time reported, and the origin status in the Azores. Sequences deposited in public repositories but not included in peer-reviewed literature are referred to as “unpublished”. Taxa marked with “*” represent updates for the Azorean marine flora.
TaxaGenetic MarkersFirst Report to the AzoresStatus in the AzoresComments
Phylum Ochcrophyta
  Class Phaeophyceae
    Order Dictyotales
      Family Dictyotaceae
Dictyota cyanoloma
Tronholm, De Clerck, A.Gómez-Garreta & Rull Lluch		psbA
[24,25]		2006
[24]		Native
[7]
Dictyota dichotoma
(Hudson) J.V.Lamouroux		psbA; cox1
[24,25]		1928
[26]		Native
[7]
Dictyota implexa
(Desfontaines) J.V.Lamouroux		psbA; cox1
[24,25]		1870
[27]		Native
[7]
Dictyota pleiacantha
Tronholm *		psbA
[25]		2005
[25]		Native
(This study)		Phylogenetic reconstruction of Dictyota pleiacantha based on multiple genes (psaA, psbA, rbcL, cox1; [28]) indicates that the species comprises two clades, one occurring in the Canary Islands (type locality), Madeira and the Azores, therefore a Macaronesian endemic, and another occurring on the Western Atlantic coasts.
Lobophora delicata
Camacho & Fredericq		cox3
[28]		2018
[28]		Native
(This study)		According to Vieira et al. [25,29], the Azores are within the species’ native distributional range.
Padina gymnospora
(Kützing) Sonder *		UPA
(This study)		First report
(This study)		Cryptogenic
(This study)		The species currently has a worldwide distribution and is not reported as introduced or invasive elsewhere.
Rugulopteryx okamurae
(E.Y.Dawson) I.K.Hwang, W.J.Lee & H.S.Kim		psbA, rbcL
[4]		2019
[4]		Introduced
[4]		This species exhibits a highly invasive behavior in the Azores [5,30].
    Order Ectocarpales
      Family Scytosiphonaceae
Colpomenia sinuosa
(Mertens ex Roth) Derbès & Solier		cox1, rbcL, UPA
(Unpublished; this study)		1894
[29]		Native
[7]
Hydroclathrus tilesii
(Endlicher) Santiañez & M.J.Wynne		psaA, cox3
([31]; as
Hydroclathrus stephanosorus)		2016
[31]		Introduced
[2]
Petalonia binghamiae
(J.Agardh) K.L.Vinogradova		cox1
(Unpublished)		1989
([32]; as Endarachne binghamiae)		Introduced
[2]
Scytosiphon lomentaria
(Lyngbye) Link, nom. cons.		cox1, UPA
(Unpublished; this study)		1911
([33]; as
Scytosiphon
lomentarius)		Native
[7]		Although the binomial Scytosiphon simplicissimus has priority over Scytosiphon lomentaria, the latter was maintained due to its widespread and consistent use for the species [34].
Zonaria tournefortii
(J.V.Lamouroux) Montagne		cox1
(Unpublished; [25])		1928
[26]		Native
[7]
    Order Fucales
      Family Fucaceae
Fucus spiralis
Linnaeus		cox1, UPA
(Unpublished; this study)		1911
([33]; as Fucus platycarpus var. spiralis)		Cryptogenic
[7]
      Family Sargassaceae
Cystoseira pustulata
(Ercegovic) Neiva & Serrão *		cox1, cox3, nad1, psaA
(Unpublished; [35,36])		1986
([37]; as
Cystoseira
compressa and
C. humilis)		Native
(This study)		According to Neiva et al. [36], Cystoseira compressa and C. humilis are apparently absent in the Azores and should be referred to as C. pustulatus.
According to Neiva et al. [36], the Azores are within the native distributional range of Cystoseira pustulata.
Sequences not found in the search described in Section 2.3 for missing the locality in GenBank were added to the list by the authors who knew the article where the sequences were published [35].
Ericaria selaginoides
(Linnaeus) Molinari & Guiry		cox1
[36]		1986
([37]; as
Cystoseira
tamariscifolia)		Native
([38]; as
Cystoseira
tamariscifolia)
Gongolaria abies-marina
(S.G.Gmelin) Kuntze		cox1, cox3, nad1, psaA
(Unpublished; [36])		1938
([39];
as Cystoseira abies-marina)		Native
[7]
Sargassum cymosum
C.Agardh		cox1
(Unpublished)		1986
[37]		Native
[7]
    Order Ralfsiales
      Family Pseudoralfsiaceae
Pseudoralfsia azorica
Parente, Fletcher & G.W.Saunders *		cox1, rbcL
[40]		2009
[40]		Endemic
[40]
    Order Sphacelariales
      Family Stypocaulaceae
Halopteris filicina
(Grateloup) Kützing		cox1
(Unpublished)		1938
([39];
as Sphacelaria filicina)		Native
[7]
Halopteris scoparia
(Linnaeus) Sauvageau		LSU
(Unpublished)		1911
[33]		Native
[7]
Phylum Rhodophyta
  Class Florideophyceae
    Order Bonnemaisoniales
      Family Bonnemaisoniaceae
Asparagopsis armata
Harvey		cox1
(Unpublished)		1988
[41]		Introduced
[2]		Previous reports Asparagopsis armata (see list from [42]) are based on A. delilei, which is currently a synonym of A. taxiformis.
Asparagopsis taxiformis
(Delile) Trevisan		cox1, cox2, LSU, rbcL-rbcS spacer
(Unpublished; [43])		1928
([44]; as
Asparagopsis
delilei)		Introduced
[2]
    Order Ceramiales
      Family Rhodomelaceae
Herposiphonia tenella
(C.Agardh) Ambronn		rbcL
[3]		2018
[3]		Native
[3]
Laurencia pyramidalis
Bory ex Kützing		cox1, rbcL, UPA
([45]; This study)		2011
[45]		Native
[7]
Laurencia viridis
Gil-Rodríguez & Haroun		cox1, rbcL
[45]		1981
[46]		Native
[38]
Laurenciella marilzae
(Gil-Rodríguez, Sentíes, Díaz-Larrea, Cassano & M.T.Fujii) Gil-Rodríguez, Sentíes, Díaz-Larrea, Cassano & M.T.Fujii		cox1, rbcL
[45]		2011
[45]		Native
[7]
Lophocladia trichoclados
(C.Agardh) F.Schmitz		rbcL
[3]		2018
[3]		Native
[3]
Lophosiphonia simplicissima
Díaz-Tapia		rbcL
[3]		2018
[3]		Native
[3]
Melanothamnus macaronesicus
Rodríguez-Buján & Díaz-Tapia		rbcL
[47]		2018
[47]		Native
(This study)		According to Rodriguez-Bujan et al. [47], Melanothamnus macaronesicus was only found in the Canary Islands and the Azores; therefore, it is a Macaronesian endemic.
Melanothamnus pseudoforcipatus
Díaz-Tapia		rbcL
[3]		2018
[3]		Cryptogenic
[3]
Osmundea oederi
(Gunnerus) G.Furnari		cox1, rbcL
[48]		2010
[45,48]		Native
[7]
Osmundea pinnatifida
(Hudson) Stackhouse		cox1, rbcL
[45]		1938
([39]; as Chondria pinnatifida)		Native
[7]
Polysiphonia villum
J.Agardh		rbcL
[3]		2018
[3]		Native
[3]
Vertebrata barbarae
Muñoz-Luque & Díaz-Tapia		rbcL
[49]		2018
[49]		Endemic
[49]		Sequences not found in the search described in Section 2.3 for missing the locality in GenBank were added to the list by the authors who knew the article where the sequences were published [49].
Vertebrata cymatophila
(Børgesen) Muñoz-Luque		rbcL
[49]		2018
[49]		Native
(This study)		According to Díaz-Tapia et al. [49], Vertebrata cymatophila was only found in the Canary Islands and the Azores; therefore, it is a Macaronesian endemic.
Sequences not found in the search described in Section 2.3 for missing the locality in GenBank were added to the list by the authors who knew the article where the sequences were published [49].
    Order Corallinales
      Family Corallinaceae
Corallina berteroi
Montagne ex Kützing		cox1, rbcL
(Unpublished; [50]; as
Coralllina
caespitosa)		2013
[51]		Native
[7]		Sequences not found in the search described in Section 2.3 for missing the locality in GenBank were added to the list by the authors who knew the article where the sequences were published [50].
Jania pedunculata var. adhaerens
(J.V.Lamouroux) A.S.Harvey, Woelkerling & Reviers		cox1, UPA
(This study)		1986
([37]; as Jania adhaerens)		Native
[7]		Studies based on lectotype material have supported the recognition of Jania pedunculata var. adhaerens as a variety rather than a distinct species [52]. However, the synonym Jania adhaerens is still widely used.
Jania verrucosa
J.V.Lamouroux, nom. rejic.		cox1, rbcL, UPA
(This study)		1993
[53]		Native
[7]		Although studies based on lectotype material have indicated that Jania verrucosa is a name of uncertain nomenclatural application [54], it is still widely used.
      Family Lithophyllaceae
Amphiroa beauvoisii
J.V.Lamouroux		cox1, UPA
(This study)		1886
([55]; as Amphiroa exilis)		Native
[7]		Gain [33] is the first to report Amphiroa beauvoisii to the Azores based on Piccone [55]. Nevertheless, Piccone reported A. exilis (currently a valid species) with various synonyms, including A. pustulata. (currently a synonym of A. beauvoisii). Although it is unknown how or when the species identity was clarified, the date of the original record was maintained.
    Order Gelidiales
      Family Gelidiaceae
Gelidium microdon
Kützing		rbcL, UPA
(This study)		1938
([39]; as Sphaerococcus corneus var. spinulosus)		Native
[7]
    Order Gelidiales
      Family Pterocladiaceae
Pterocladiella capillacea
(S.G.Gmelin) Santelices & Hommersand		cox1
(Unpublished)		1886
([55]; as Pterocladia capillacea		Native
[7]
    Order Gigartinales
      Family Cystocloniaceae
Hypnea musciformis
(Wulfen) J.V.Lamouroux		cox1, rbcL, UPA
(This study)		1911
[33]		Cryptogenic
[7]
    Order Nemastomatales
      Family Nemastomataceae
Predaea feldmannii subsp. azorica
Gabriel		rbcL
[56]		1990
([57]; as Predaea feldmannii)		Endemic
[56]
      Family Schizymeniaceae
Platoma cyclocolpum
(Montagne) F.Schmitz		rbcL
[58,59]		1986
[37]		Native
[7]
Schizymenia apoda
(J.Agardh) J.Agardh		LSU, rbcL
[59,60,61]		1894
([29]; as Schizymenia obovata and S. undulata)		Native
[7]
    Order Peyssonneliales
      Family Peyssonneliaceae
Olokunia boudouresquei
(Yoneshigue) Pestana, Lyra, Cassano & J.M.C.Nunes *		rbcL
(Unpublished; as Cruoriopsis crucialis)		First report
(This study)		Cryptogenic
(This study)		Olokunia boudouresquei is mostly an Atlantic species, with few reports to Australia and New Zealand. Due to the taxonomic confusion within Peyssonneliaceae [62,63], further studies are necessary to assess the species status in the Azores.
Peyssonnelia coriacea
Feldmann		LSU, rbcL
([60,64]; as Peyssonnelia squamaria)		1990
[65]		Native
([7]; as Peyssonnelia squamaria)		Reappraisal of sequences of Peyssonnelia species indicates that the species reported from the Azores as P. squamata (KR732900) may be P. coriacea [62,63]. The sequencing of type material is necessary to confirm the new identity.
    Order Rhodymeniales
      Family Lomentariaceae
Lomentaria articulata
(Hudson) Lyngbye		cox1, UPA
(This study)		1911
[33]		Native
[7]
Phylum Chlorophyta
  Class Ulvophyceae
    Order Bryopsidales
      Family Caulerpaceae
Caulerpa prolifera
(Forsskål) J.V.Lamouroux		tufA
[66]		2013
[67]		Introduced
[2]
Caulerpa webbiana f. disticha
Vickers		tufA
(Unpublished)		2002
([68]; as Caulerpa webbiana)		Introduced
([68]; as Caulerpa webbiana)		This species has an invasive behavior in the Azores [68,69].
      Family Codiaceae
Codium adhaerens
C.Agardh		rpl16, rbcL
[70]		1912
[33]		Native
[7]
    Order Ulvales
      Family Ulvaceae
Ulva australis
Areschoug *		tufA
(This study)		First report
(This study)		Introduced
(This study)		Molecular analyses point to a greater genetic diversity of Ulva australis in the Northwest Pacific, with populations in Australia, New Zealand, California, Chile, and Europe deriving from multiple introductions [71,72,73]. In European waters, Ulva australis was discovered at several scattered locations in the 1990s and early 2000s, including the Delta region of the Netherlands, the Dutch Wadden Sea [74], off Brittany [75], the Iberian Peninsula, from the Bay of Biscay to Portugal [76,77] and the Mediterranean Sea (e.g., [78,79,80]). Possible introduction vectors include ballast water, hull fouling, and oyster transplants.
Ulva compressa
Linnaeus		tufA
(This study)		1938
[39]		Native
[7]
Ulva lactucarbcL
([81]; as Ulva fasciata)		1938
[39]		Cryptogenic
[7]		Loughane et al. [81] provided an rbcL sequence (EU484418) of a voucher identified as Ulva fasciata. Later studies including type material [82] corrected the voucher identity to Ulva lactuca.
Ulva lacinulata
(Kützing) Wittrock *		tufA
(This study)		First report
(This study)		Cryptogenic
(This study)		According to Hughey et al. [83], there is a common taxonomic confusion with this species, which has been mistakenly reported as U. laetevirens (JQ048945), U. scandinavica (MW570777), U. armoricana (AB097632), and U. rigida (EU484395). Genetic investigations of Ulva spp. in the Baltic Sea and the Atlantic proximities showed that U. rigida was absent from the studied areas while U. lacinulata was relatively frequent [84]. The worldwide distribution of Ulva species needs to be reappraised based on sequences from type material due to the general misuse of species names [85]. Additionally, further studies are necessary to assess the real identity of U. rigida, which is currently reported to be native to the Azores [7].
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Gabriel, D.; Schmidt, W.E.; Micael, J.; Moura, M.; Fredericq, S. DNA Barcode-Assisted Inventory of the Marine Macroalgae from the Azores, Including New Records. Phycology 2024, 4, 65-86. https://0-doi-org.brum.beds.ac.uk/10.3390/phycology4010004

AMA Style

Gabriel D, Schmidt WE, Micael J, Moura M, Fredericq S. DNA Barcode-Assisted Inventory of the Marine Macroalgae from the Azores, Including New Records. Phycology. 2024; 4(1):65-86. https://0-doi-org.brum.beds.ac.uk/10.3390/phycology4010004

Chicago/Turabian Style

Gabriel, Daniela, William E. Schmidt, Joana Micael, Mónica Moura, and Suzanne Fredericq. 2024. "DNA Barcode-Assisted Inventory of the Marine Macroalgae from the Azores, Including New Records" Phycology 4, no. 1: 65-86. https://0-doi-org.brum.beds.ac.uk/10.3390/phycology4010004

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