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Article

DNA Barcoding of Red Algae from Bocas del Toro, Panamá, with a Description of Gracilaria bocatorensis sp. nov. and G. dreckmannii sp. nov. (Gracilariales, Gracilariaceae)

by
Maycol Ezequiel Madrid Concepcion
1,*,
Rachel Collin
2,
Kenneth S. Macdonald III
3,
Amy C. Driskell
3,
Suzanne Fredericq
4,
Brian Wysor
5 and
D. Wilson Freshwater
6
1
Biology and Marine Biology Department, University of North Carolina Wilmington, Wilmington, NC 28403, USA
2
Smithsonian Tropical Research Institute, Ancon, Panama 0843, Panama
3
Laboratories of Analytical Biology, National Museum of Natural History, Smithsonian Institution, Washington, DC 20560, USA
4
Department of Biology, University of Louisiana at Lafayette, Lafayette, LA 70504, USA
5
Department of Biology, Marine Biology & Environmental Science, Roger Williams University, Bristol, RI 02809, USA
6
Center for Marine Science, University of North Carolina Wilmington, Wilmington, NC 28409, USA
*
Author to whom correspondence should be addressed.
Diversity 2025, 17(4), 222; https://doi.org/10.3390/d17040222
Submission received: 20 February 2025 / Revised: 15 March 2025 / Accepted: 20 March 2025 / Published: 23 March 2025
(This article belongs to the Special Issue DNA Barcodes for Evolution and Biodiversity—2nd Edition)
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Abstract

:
Bocas del Toro is an archipelago on the Caribbean coast of Panamá, recognized as a biodiversity hotspot. While marine red macroalgae in the Western Atlantic are well studied, the marine flora of Panamá, particularly Bocas del Toro, remains underexplored using DNA barcoding. This study documents the diversity of marine red macroalgae in the region using COI-5P barcoding to identify species, detect cryptic diversity, and assess the presence of invasive and amphi-isthmian species. Specimens collected in 2008 and 2009 yielded 179 COI-5P sequences. Barcode Index Numbers (BINs) were assigned to 82 genetic clusters, many lacking GenBank matches, suggesting potential new species. Morphology and phylogenetic analyses of rbcL, UPA, and cox1 confirmed two new species of Gracilaria (G. bocatorensis sp. nov. and G. dreckmannii sp. nov.). Despite advances in DNA barcoding, red macroalgal diversity in Panamá remains understudied, particularly Corallinales, where rbcL sequences are lacking. No introduced or amphi-isthmian species were detected. This study adds 16 new species records for the Caribbean coast of Panamá, emphasizing the importance of DNA barcoding in biodiversity research.

1. Introduction

The Bocas del Toro Archipelago is a small island system located on the Caribbean coast of Panamá, in Central America. This archipelago has been extensively studied thanks to a remote scientific research station of the Smithsonian Institution that each year serves dozens of scientists from all around the world [1,2,3,4,5,6]. Since its construction, this area has been the focus of major biodiversity inventories for several marine organisms and land plants [7,8,9,10,11,12,13,14,15,16,17,18]. Marine macroalgae of the Caribbean coast of Panamá have been extensively studied in floristic inventories and ecological assessments [19,20,21,22,23,24,25,26,27]. Despite the extensive phycological surveys carried out in the area, there is very little published information about DNA barcoding of the macroalgae. Earlier collections did not preserve material for DNA extraction, but since 2007, four field phycology workshops and additional phycological expeditions have been based at the Bocas Research Station and as a result, thousands of specimens have been collected and preserved for future studies. However, only studies including a small portion of these specimens have been published [28,29,30,31,32,33,34,35,36,37,38,39].
Recent checklists of macroalgae in the Panamanian Caribbean region have focused on brown and green algae [26,27,40,41], and the cited sources for these checklists identified specimens based on morphology. Although the checklists do not include red algae, many of the cited sources do, and these records are compiled in the Smithsonian Tropical Research Institute (STRI)—Symbiota database [42]. This database currently includes reports for 146 species of red algae in the Bocas del Toro area and 227 for the whole Panamanian Caribbean, most of which have only been identified using morphological characters.
The use of morphological characters in red algae identification can be troublesome due to the plasticity of some species and high levels of cryptic diversity in the group [43,44,45,46,47]. It is necessary to complement morphological identifications with data from DNA sequences for these reasons. Several barcoding initiatives have been carried out in red algae over the last two decades, providing a rich database of DNA barcodes that can be used to identify species [48,49,50]. These efforts have been greatly improved by the sequencing of historical type specimens, which provides DNA data that can be used to tie old names to newly collected specimens [51,52,53,54,55,56].
Most of the DNA barcodes from Bocas del Toro were part of major worldwide phylogenetic studies that were published between 2003 and 2024 [32,39,57,58,59,60,61]. The number of released sequences is less than 160, with most of these belonging to the Rhodomelaceae tribes Polysiphonieae and Streblocladieae [28], crustose coralline algae (CCA) [31,32,35,36,60,61,62], and Gelidiales [29,30,33]. These studies have shown that this archipelago presents a unique diversity of species and that many generated sequences were from species new to DNA sequence repositories. Anthropogenic impacts are increasing in the Archipelago of Bocas del Toro because of the constant development of residential areas [63,64,65] and recent introductions of seaweed farms [66,67]. Panamá is also a major waterway for shipping traffic, which can led to biological invasions [68], so for these reasons it is crucial to develop a baseline of the rich diversity of red algae in the region [6].
The main objective of this study was to inventory the red algae of the Bocas del Toro Archipelago using DNA barcoding in order to establish a detailed and DNA sequence-verified list of species for the region. In addition, using these data the diversity of Gracilaria along the Caribbean coast of Panamá was clarified and two new species are described.

2. Materials and Methods

2.1. Sample Collection and Processing

Samples of red algae were collected from fourteen sites (Figure 1) around the Bocas del Toro Archipelago during the 2008 Tropical Field Phycology and 2009 NSF-PASI Advanced Tropical Phycology workshops (https://striresearch.si.edu/taxonomy-training/past-courses/ (accessed on 25 December 2024)). Snippets from 215 specimens were sampled and examined under a stereoscope; epiphytes were carefully removed, and tissue samples were dried completely for 2–3 days with fresh silica gel.

2.2. DNA Extraction, Amplification, and Sequencing

DNA was extracted from dried tissue samples using a highly modified AutoGenprep 965 extraction robot procedure (AutoGen Inc., Holliston, MA, USA). A small, dried tissue section (<1 mm) was removed from each dried sample. Samples were removed from silica, added to 50 µL 100% EtOH, and then bead-beat on a TissueLyser (QIAGEN, Germantown, MD, USA) at max speed for 30 s. Next, 500 µL Lysis buffer (Qiagen DNeasy Plant Kit buffer AP1—Cat. No. 1014630) was added, followed by an overnight digestion at 56 °C. After digestion, 150 µL neutralization buffer (Qiagen DNeasy Plant Kit either AP2 or P3) was added. Following centrifugation for 20 min @ 2100× g, 300 µL supernatant was removed and extracted on the AutoGenprep 965 extraction robot using the Mouse Tail protocol setting. Sequences of the Cytochrome c Oxidase Subunit I 5′ region (COI-5, 664 base pairs, bp) were amplified using the primer pair GazF1 and GazR1 [48], and when those primers failed to amplify, GWSLF3 and GWSRx [69] were used. The 10 μL PCR cocktail for COI-5P included 5 μL GoTaq Hot Start Mix (Promega, Madison, WI, USA), 0.1 μL BSA, and 0.3 μL of each 10 mM primer. Thermocycler profiles for amplification used an initial denaturation at 95 °C for 7 min, followed by 40 cycles of 95 °C for 30 s, 45 °C for 30 s, and 72 °C for 45 s, finishing with a final 90 s step at 72 °C. DNA for the amplification and sequencing of domain V of the 23S plastid rRNA gene or Universal Plastid Amplicon (UPA, 370 bp) and the plastid gene for ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL, 1387 bp) was extracted from Gracilaria duplicate samples during 2024 using the Meridian MyTaq™ Extract-PCR Kit. Amplification and sequencing were performed using the UPA primers p23SrV_f1 and p23SrV_r1 [70], rbcL primers F57, F753 [71], F1210 [72], F505, F808, R766, R1111, R1144, R1444 [73], and following previously described methods [52,74]. Some DNA extracted in 2024 was fragmented and had to be amplified by short fragments using the following primer combinations: F57-R766, F753-R1111, F505-R1144, F808-R1444, F808-RrbcSStart, and F1210-R1444. Amplified PCR products were cleaned using Applied Biosystems™ ExoSAP-IT (Thermo Fisher Scientific Inc., Waltham, MA, USA). Sequencing of COI-5P was performed using the BigDye kit (Thermo Fisher Scientific Inc.) or BrilliantDye™ Terminator v3.1 Cycle Sequencing Kit (NimaGen, Nijmegen, The Netherlands) for the rbcL and UPA. Sequence reactions were run in house on an ABI3500 Series Genetic Analyzer (Thermo Fisher Scientific Inc.).
Forward and reverse chromatograms were subsequently aligned, and primer sequences were trimmed using Geneious Prime version 2025.0.3 (Dotmatics, Boston, MA, USA). The newly generated sequences were uploaded to BOLD Systems (https://bench.boldsystems.org/index.php (accessed on 25 December 2024), Centre for Biodiversity Genomics, University of Guelph, Guelph, ON, Canada), where they were clustered into Barcode Index Numbers (BINs). FASTA-formatted sequences were then downloaded from BOLD and aligned using MUSCLE in Geneious Prime. UPGMA trees and pairwise distances were calculated using the raw p-distance method in MEGA v11 [75].

2.3. Sequence Identification and Phylogenetic Analysis

NCBI batch BLAST searches (https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 25 December 2024)) were performed for all sequences, and the top hits based on percent identity were recorded for each BIN. Phylogenetic analyses of sequences from the order Gracilariales were conducted using both maximum likelihood (RAxML) and Bayesian methods. For these analyses, an initial RAxML analysis was conducted using all 1508 rbcL and 2354 Cytochrome c Oxidase Subunit I (cox1) or COI-5P sequences from GenBank. Maximum likelihood analysis was performed in Geneious Prime using RAxML with the GTR + I + G substitution model and 1000 rapid bootstrap replicates, followed by a search for the best-scoring tree [76]. One representative sequence from each Gracilariales species resolved in this initial analysis was used in subsequent analyses. Priority for selecting the representative sequence was given first to a type sequence, followed by a sequence from the type locality, and lastly, the longest available sequence. If the type sequence was short (less than 200 bp for rbcL or 400 bp for cox1), an additional longer sequence of that species was also included in the analyses. After choosing one representative per species, a maximum likelihood analysis was performed with the newly generated sequences, as previously mentioned [76]. Bayesian inference was conducted with MrBayes in Geneious using MCMC analysis for 10 million generations, sampling every 1000 generations [77]. A 50% majority-rule consensus tree was constructed from two independent MCMC runs after discarding the first 20% of generations as burn-in, and posterior probabilities were calculated. Species delimitations for the Gracilaria cervicornis clade were made using the automatic barcode gap discovery (ABGD) method and Multi-rate Poisson tree processes (mPTPs). The ABGD analysis was conducted in the web interface (https://bioinfo.mnhn.fr/abi/public/abgd/ (accessed on 25 December 2024)) using K2P distances for the UPA and simple distance (p-distances) for rbcL and cox1 [78]. The remaining options were left with the default settings (0.001 Pmin, 0.1 Pmax, 10 steps, and 1.5 X gap width). For the mPTP, we made a RAxML tree using the methods previously described, and we uploaded it on the web version of the tool (https://mcmc-mptp.h-its.org/ (accessed on 25 December 2024)), where we used the mPTP model (multi), a null model starting delimitation, and a 0.0001 minimum branch length. MCMC settings were 1000 burnin, sampling every 1000, and 1 million steps [79].
We followed the revised generic concepts of the Gracilariaceae by Gurgel et al. [59,80,81,82], which recognized three monophyletic genera previously classified under Gracilaria sensu lato. These genera were found to be systematically valid based on both the comparative morphological development of male structures and postfertilization stages, as well as the molecular-based phylogenetic topology of the family. The genera include Agarophyton (A. tenuistipitata, A. vermiculophylla, and A. chilensis), Crassiphycus (C. usneoides, C. crassisimus, C. corneus, C. birdiae, C. secundus, C. changii, C. punctatus, C. caudatus, “G.” cliftonii, and “G.” truncata) and Hydropuntia (H. urvillei, H. eucheumatoides, H. multifurcata, H. rangiferina, H. edulis, H. perplexa, and H. preissiana). This taxonomic decision is in contrast with the subsequent paper by Lyra et al. [83], who subsumed these three genera back into Gracilaria. The genus Gracilariopsis is not addressed in this paper.

2.4. Morphological Analysis

Morphological identification of all specimens was conducted during the 2008–2009 workshops using the available keys at the time [24,84]. For these identifications, we prepared cross-sections and whole mounts from fresh material to verify species under the microscope. Morphological characteristics of the new Gracilaria species were documented in 2024 using dried specimens preserved in silica gel. Hand sections of the dried specimens were rehydrated with distilled soapy water, stained with aniline blue, and then mounted in Karo corn syrup for microscopic examination. Photographs were taken using an Olympus BX41 microscope connected to an Olympus OM-D E-M5 Mark II camera (Olympus, Tokyo, Japan). Voucher specimens were deposited at the David J. Sieren Herbarium (WNC) at the University of North Carolina Wilmington; Max & Fran Hommersand Algae Herbarium: Algae (NCU) at the University of North Carolina at Chapel Hill; and the Herbarium of the University of Panamá (PMA). Herbarium voucher acronyms follow Index Herbariorum [85] and the validity of species names was verified in AlgaeBase [86].

3. Results

3.1. Barcode Analyses

We obtained a total of 179 COI-5P sequences, of which 111 were unique (Table 1). Of the 179 sequences, 160 were 664 bp long, covering the full length of the amplifiable region targeted by the primers. Nineteen sequences ranged from 551 to 663 bp in length. The BOLD SYSTEMS workbench clustered the 179 COI-5P sequences into 82 Barcode Index Numbers (BINs) that represent putative species (Figure 2). The intraspecific divergence between these 82 BINs ranged from 0% to 1.129%, while the interspecific distances ranged from 2.711% to 23.644% (Figure 3). A barcode gap between 1.13% and 2.71% divergence was found in the COI-5P sequences from Bocas del Toro (Figure 2 and Figure 3).
The BLAST searches showed that more than half of the BINs (47 out of 82) lacked close similarity (BLAST percentage identity below 97.29%) to available sequences in GenBank (Figure 4 and Table 1) and represent either new species or known species for which no COI-5P sequences are in GenBank. The minimum BLAST percentage similarity was 85.7%, and all the BINs were assigned to eight orders of red algae from the class Florideophyceae (Corallinales, Gracilariales, Ceramiales, Rhodymeniales, Nemaliales, Gelidiales, Halymeniales, and Peyssonneliales). The orders Ceramiales, Corallinales, and Gracilariales had the highest number of BINs, 21, 18, and 14, respectively. Sixteen BINs are new species records for the Caribbean coast of Panamá, and twenty-four are from Western Atlantic species that currently do not have a COI-5P (Table 1). BINs from the order Corallinales had the highest number of new BINs not represented on GenBank or BOLD SYSTEMS (13 out of 18), but due to the lack of rbcL sequences we cannot confirm whether these BINs represent new species or known species that currently lack COI-5P sequences (Table 1). In contrast, BINs from the order Gracilariales mostly had high BLAST percentage similarity to known species in GenBank, and only 2 out of 14 BINs had a low percentage of similarity (Table 1). The morphological characteristics of the vegetative habit of one BIN (BOLD:ADB6885) resembled species described from the Pacific coast of Panama, such as Gracilaria parva, as well as the Caribbean species G. galetensis and G. gurgelii. In contrast, the other BIN (BOLD:ACW4300) had a cylindrical thallus similar to several species, including G. microcarpa and G. apiculata. We obtained rbcL and UPA sequences for the samples of these two BINs and found no close homology between them and the many Tropical Western Atlantic and Caribbean Sea Gracilaria species for which these loci have been generated. Herein, we proceed to formally describe them as Gracilaria bocatorensis sp. nov. (BOLD:ADB6885) and Gracilaria dreckmannii sp. nov. (BOLD:ACW4300).

3.2. Gracilaria Specific Sequence Analyses and New Species Support

The rbcL RAxML and Bayesian phylogeny of the genus Gracilaria resolved G. dreckmannii sp. nov. within a highly supported clade with 93% bootstrap support (BS) and 0.95 posterior probability (PP) that includes six species from the Tropical Western Atlantic (G. mammillaris, G. cuneata, G. microcarpa, G. apiculata, G. ferox, and G. cervicornis) (Figure 5). Within this clade, G. microcarpa was identified as the sister species to G. dreckmannii sp. nov. (see description below) with full support (100% BS, 1.00 PP). In the same rbcL phylogeny, G. bocatorensis sp. nov. (dee description below) formed a highly supported clade (96% BS, 0.99 PP) with three Atlantic species (G. gurgelii, G. galetensis, and Gracilaria sp. NC, USA) and one species from the Tropical Eastern Pacific (G. parva). G. bocatorensis sp. nov. was resolved as the sister species to G. parva, with strong support (99% BS, 0.99 PP) (Figure 5). Additionally, we obtained an rbcL sequence from one sample (ABBAD061-15; TFP08-131; WNC34522) that was identical to the rbcL sequence of the holotype of G. suzanneae from Brazil (Figure 5).
The cox1 RAxML and Bayesian phylogeny resolved G. dreckmannii sp. nov. within a clade containing the same species as the rbcL phylogeny, apart from G. apiculate, for which there is no cox1 sequence; however, this clade had low support (BS < 70%, PP < 0.90) (Figure 6). Additionally, G. dreckmannii and G. microcarpa were not recovered as sister species, but there was no support for their relationships within the clade. The cox1 phylogeny produced results for G. bocatorensis similar to those of the rbcL phylogeny, with the only difference being the absence of cox1 sequences for G. galetensis and G. gurgelii (Figure 6). We also generated UPA sequences for G. dreckmannii and G. bocatorensis. However, this dataset lacked sequences for several species within the clades containing G. dreckmannii sp. nov. and G. bocatorensis sp. nov., making it currently impossible to perform comparisons similar to those made with the rbcL and cox1 datasets.
Inconsistencies were found in the identification of G. cervicornis and G. mammillaris specimens in past studies [87,88], two species which are closely related to G. dreckmannii sp. nov. (Figure 7, Figure 8, Figure 9 and Figure 10). A recent revision clarified that G. cervicornis and G. ferox are distinct species and placed rbcL sequences of four specimens previously labeled as G. cervicornis (GenBank accessions AY049366, AY049368, AY049367, and AY049365) in G. ferox [87]. These four specimens were later shown to be closely related (0.1% to 0.4% pairwise distance) to G. microcarpa [88], and subsequent studies confirmed that these four rbcL sequences were unrelated to rbcL sequences in the plastid genomes of G. ferox (GenBank accessions MH396010 and MZ336058) [83,89], indicating that they belong to G. microcarpa (Figure 7 and Figure 8). The nine currently available rbcL sequences labeled as G. ferox and G. cervicornis formed a fully supported clade (100% BS, 1.00 PP) (Figure 8). Species delimitation analyses indicated that they represent the same species (Figure 7 and Figure 8), which was sister to Gracilaria apiculata (98% BS, 1.00 PP) (Figure 8). Nuclear gene phylogenomic analyses revealed non-monophyly among G. ferox and G. cervicornis specimens, further supporting this conclusion [83]. Additionally, two rbcL sequences (vouchers ALCB103145 and ALCB103175; GenBank accessions KP210218 and KP210219) formed a clade with other G. ferox sequences (62% BS, 0.71 PP) (Figure 8), but the cox1 sequences of the same individuals (GenBank accessions KT353011 and KP210156) grouped with G. cervicornis (56% BS, 0.63 PP) (Figure 9). Based on this evidence, we conclude that G. ferox and G. cervicornis are the same species, and the name G. cervicornis has priority. However, we are not formally proposing this synonymy in this manuscript, as we believe the validity of the names should be maintained until DNA sequences from the type specimens are obtained.
A similar issue was observed among G. mammillaris, G. curtissiae, and G. cuneata. Phylogenetic and species delimitation analyses of rbcL and cox1 indicated that all sequences identified as these three species belong to a single species (Figure 5, Figure 6, Figure 7, Figure 8 and Figure 9). One rbcL sequence (voucher ALCB65130; GenBank accession MW723504) formed a clade with G. cuneata (88% BS, 0.61 PP) (Figure 8), but in the UPA phylogeny it formed a clade with G. mammillaris (70% BS, 0.77 PP) (Figure 10). Similarly, a cox1 sequence of G. cuneata (voucher ALCB97567; GenBank accession KP21016592) also grouped with G. mammillaris in the UPA phylogeny (Figure 9 and Figure 10). Species delimitation analyses and inconsistencies in sequences from the same individuals strongly suggest that these three names represent the same species. Since G. mammillaris is the only one with a holotype sequence (GenBank accession MH01729964) [90], we propose using this name. Species name synonymizing will not be performed until DNA sequences are generated from the holotype specimens of the species within these two groups. The UPA sequences yielded inconsistent results in the species delimitation analyses (Figure 7 and Figure 10). Distance-based methods (ABGD) identified a separate species for every unique sequence, even with just a single base pair difference, whereas the mPTP method grouped all sequences into a single species (Figure 7 and Figure 10). While the UPA has been shown to be adequate for some red algae, these discrepancies suggest its limitations for delimiting species in this study.
With the proposed changes, the clade of G. dreckmannii sp. nov. includes four accepted species (G. apiculata, G. cervicornis, G. mammillaris, and G. microcarpa) and one undescribed species from Brazil (Gracilaria sp., voucher SPF57166, field ID BRA34). The rbcL intraspecific p-distances within these six species ranged from 0% to 1.41%, while interspecific distances ranged from 1.58% to 4.76% (Table 2 and Table S1). The nearest neighbor (NN) of G. dreckmannii is G. microcarpa, with a minimum interspecific distance of 1.58% (Table 2 and Table S1). For cox1, intraspecific p-distances were higher (0% to 3.29%), as were interspecific distances (4.43% to 9.83%) (Table 2 and Table S2). G. microcarpa was also the NN of G. dreckmannii in analyses of this gene, with a minimum interspecific distance of 4.43% (Table 2 and Table S2). UPA p-distances were the smallest, ranging from 0% to 0.54% for intraspecific distances and 0.81% to 3.00% for interspecific distances (Table 2 and Table S3).
Intraspecific rbcL p-distances among the five species in the G. bocatorensis clade (G. gurgelii, G. galetensis, G. parva, Gracilaria sp., voucher WNC21012, and G. bocatorensis sp. nov.) ranged from 0% to 0.15%, while interspecific distances ranged from 1.11% to 3.03% (Table 2 and Table S4). cox1 intraspecific p-distances ranged from 0% to 1.20%, and interspecific distances ranged from 4.85% to 5.17% (Table 2 and Table S2). G. parva was the NN of G. bocatorensis sp. nov. for both genes, with minimum interspecific distances of 1.55% (rbcL) and 4.97% (cox1) (Table 2, Tables S2 and S4). Lastly, the UPA intraspecific distance was 0%, and the interspecific distances ranged from 0.81% to 1.35% (Table 2 and Table S3).

3.3. New Species Descriptions

Gracilaria bocatorensis M. Madrid and Freshwater sp. nov. Figure 11 and Figure 12.
DESCRIPTION: The thalli were pink to dark red or brown, cartilaginous to coriaceous, and grew as erect, subdichotomously to irregularly branched plants, holdfast not seen (Figure 11a,b). Main axes and branches were 2.5–7.7 cm long, 1.7–4.0 mm wide, and 328–417 µm thick, and branched up to 5 orders; terete to compressed near the base and otherwise compressed to flattened with entire margins and obtuse apices (Figure 11a–d). The medulla was composed of 4–8 layers of ovoid to globose medullary cells that were 56–102 µm in diameter (Figure 11c,d). The cortex included 1–3 layers of quadrate cells, 2.7–10 µm in diameter (Figure 11e). There was an abrupt transition between the medulla and cortex in the apical portion of the thallus (Figure 11c,d).
Spermatangia and tetrasporangia were not observed. Cystocarps were present on both blade surfaces of female gametophytes, scattered throughout the thallus (Figure 11a). Mature cystocarps were hemispherical, 0.7–0.8 mm in diameter (Figure 11a). Immature cystocarps were slightly constricted around the base (Figure 12a). The cystocarp floor was composed of 3–5 layers of darkly staining oblong to stellate cells (Figure 12b). Tubular nutritive cells were not abundant, with 1–2 observed in cross-sections (Figure 12b). They were 46–73 µm long, 11–14 µm wide, and developed from the gonimoblast to the inner pericarp (Figure 12b). Outer pericarp was composed of 9–11 layers of cells that were globose or quadrate to cylindrical in the outer layers and stellate within the inner layers (Figure 12c). Secondary pit connections were present throughout the outer and inner pericarp (Figure 12b,c). Fusion cells were not observed. Gonimoblast was globose, unlobed, and formed by a dense mass of compact gonimoblast filaments (Figure 12d). Carposporangia were arranged in unbranched chains, with mature carposporangia subspherical to ovoid and 14–18 µm in diameter (Figure 12e).
GenBank accession numbers for the holotype specimen: cox1—PV136111.
HOLOTYPE: WNC34521, field ID collection number TFP08-233 (PHYKOS-5363), in the drift in the intertidal zone during low tide, Playa Istmito, Beach across from STRI-Bocas Research Station, Isla Colón, Subdistrict—Bocas del Toro, District—Bocas del Toro, State—Bocas del Toro, Country—Panamá, 9.351817 N, 82.255104 W, 0 m, 15 July 2008, leg. Suzanne Fredericq.
ISOTYPE: PMA0140436, field ID collection number TFP08-233 (PHYKOS-5363).
OTHER SPECIMENS EXAMINED: WNC34630, field ID collection number TFP08-229 (PHYKOS-5359), in the drift in the intertidal zone during low tide, Playa Istmito, Beach across from STRI-Bocas Research Station, Isla Colón, Subdistrict—Bocas del Toro, District—Bocas del Toro, State—Bocas del Toro, Country—Panamá, 9.351817 N, 82.255104 W, 0 m, 16 July 2008, leg. Suzanne Fredericq.
HABITAT: Unknown as all specimens were found in the drift on a sandy beach.
DISTRIBUTION: Gracilaria bocatorensis sp. nov. is currently only known from the Caribbean coast of Panamá. Considering the location where it was collected, it is most likely to be found in other places around the western and perhaps wider Caribbean region.
ETYMOLOGY: this species is named after Bocas del Toro, which is the name of the state, county, and municipality of the type locality.
Gracilaria dreckmannii M. Madrid and Freshwater sp. nov. Figure 13 and Figure 14.
DESCRIPTION: The thalli were pink and yellow to dark red or brown, cartilaginous to coriaceous, and grew as erect, subdichotomously to irregularly branched plants, attached to substrate by a discoid holdfast (Figure 13a–c). Main axes and branches were 2.4–4.8 cm long, 1.9–3.0 mm wide, and 660–1200 µm thick, and branched up to 7 orders; terete throughout the whole thallus with entire margins and highly branched apices (Figure 13a–c). The medulla was composed of 7–11 layers of ovoid to globose medullary cells that were 88–117 µm in diameter (Figure 13d,e). The cortex included 1–2 layers of globose or elongated cells, 3.3–9.8 µm in diameter (Figure 13f–h). There was an abrupt transition between the medulla and cortex in the apical portion of the thallus (Figure 13d,e).
Spermatangia and tetrasporangia were not observed. One mature cystocarp was observed in the apical portion of a thallus. The cystocarp was dome-shaped, 0.5 mm in diameter, and constricted at the base (Figure 14a). The cystocarp floor was composed of 1–3 layers of darkly staining elongated cells (Figure 14a,b). The outer pericarp was composed of 9–11 layers of cells that were elongated and narrow in the outer layers and stellate within the inner layers (Figure 14c). Secondary pit connections were present throughout the outer and inner pericarp (Figure 14a–c). Fusion cells were not observed. The gonimoblast was globose, unlobed, and formed by a dense mass of compact gonimoblast filaments (Figure 14a,d,e). Tubular nutritive cells were not abundant, with 1–2 observed in cross-sections (Figure 14e). They were 81–90 µm long, 4–8 µm wide, and developed from the gonimoblast to the outer pericarp (Figure 14e). Carposporangia were arranged in unbranched chains, with mature carposporangia subspherical to ovoid and 8–14 µm in diameter (Figure 14d,e).
GenBank accession numbers for the holotype specimen: rbcL—PV136121; cox1—PV136121; UPA—PV136448.
HOLOTYPE: WNC34627, field ID collection number TFP08-346 (PHYKOS-5449), growing on rock in the intertidal zone, Flat Rock Beach, Isla Colón, Subdistrict—Bocas del Toro, District—Bocas del Toro, Province—Bocas del Toro, Country—Panamá, 9.38042 N, 82.23816 W, 0 m, 17 July 2008, leg. Olga Camacho.
ISOTYPE: PMA0140437, field ID collection number TFP08-346 (PHYKOS-5449).
OTHER SPECIMENS EXAMINED: WNC34621, field ID collection number TFP08-351 (PHYKOS-5454), on rock in the intertidal zone, Flat Rock Beach, Isla Colón, Subdistrict—Bocas del Toro, District—Bocas del Toro, Province—Bocas del Toro, Country—Panamá, 9.38042 N, 82.23816 W, 0 m, 17 July 2008, leg. Jim Norris. PMA0132532 field ID collection number Madrid M 472, on rock the intertidal zone, Flat Rock Beach, Isla Colón, Subdistrict—Bocas del Toro, District—Bocas del Toro, Province—Bocas del Toro, Country—Panamá, 9.38042 N, 82.23816 W, 0 m, 24 June 2015, leg. Maycol Madrid and Holly Cronin.
HABITAT: This species is only known from the intertidal zone of one rocky beach.
DISTRIBUTION: Gracilaria dreckmannii sp. nov. is currently only known from the Caribbean coast of Panamá. Considering the location where it was collected, it is most likely to be found in other places around the western and perhaps wider Caribbean region.
ETYMOLOGY: This species name honors Dr. Kurt M. Dreckmann, a phycologist interested in the phylogenetics and biogeography of marine macroalgae who has made significant contributions to the study of the genus Gracilaria on the Atlantic and Pacific coasts of Mexico.

3.4. New Gracilaria Species Morphological Comparisons

The Caribbean coast of Panama has records of 6 species identified using DNA sequences [91], while 25 species have been recorded based on morphological characteristics [42]. This study expands the list of DNA-verified species from 5 to 16, increasing the total number of recorded species to 33. Morphological identification of species in this genus is challenging due to high levels of morphological plasticity and the presence of cryptic species. The two new species described in this study exhibit distinct growth forms: one has flat blades, while the other has a cylindrical thallus. Based on these characteristics, they can be grouped into two distinct morphological categories.
The flat-bladed new species (Gracilaria bocatorensis) closely resembles others previously described in the region, particularly those detailed by Gurgel et al. [91] (Table 3). This group includes phylogenetically related species such as G parva, G. galetensis, and G. gurgelii, as well as several species that are not closely related, such as G. oliveirarum, G. hayi, G. cearensis (syn. G. smithsoniensis), and G. occidentalis. Species identification within this group is extremely difficult due to the extensive overlap of vegetative characteristics among all the species (Table 3). The cylindrical new species (Gracilaria dreckmannii) also resembles several cylindrical species from the region that are closely related phylogenetically, like G. microcarpa and G. apiculata. The other species that are within the same clade (G. ferox, G. cervicornis, G. cuneata, and G. mammilaris) are not similar to G. dreckmannii since they are flat blades (Table 4).

4. Discussion

The results of this study highlight the previously undocumented rich diversity of marine red algae, building upon findings from earlier research conducted in the Bocas del Toro Archipelago [7,97,98,99]. We generated 179 COI-5P, 5 rbcL, and 5 UPA sequences from 179 samples, effectively doubling the number of publicly available red algae sequences from the Caribbean coast of Panamá in GenBank (previously 152 sequences from 93 samples) (Table S5). With these new data, we increased the number of DNA-verified red algal genetic operational taxonomic units from the Caribbean coast of Panamá from 57 (Table S5) to 126. Some of these new records expand the species distributions to the Caribbean region, such as Gracilaria baiana, G. suzanneae, Gelidiella flabella, and Hypnea wynnei. This study represents the largest DNA barcoding study targeting a diverse number of red algae species from Panamá. The first study [100], conducted on the Pacific coast of Panamá at Punta Burica, analyzed 45 red algae specimens and identified 26 species, 21 of which were new records for Pacific Panamá. These findings reveal that Panamá hosts a rich marine flora along both coasts and that DNA barcoding is a helpful tool for elucidating that species richness.
Our findings also reveal areas of molecular diversity where there is a strong understanding of the current species of red macroalgae in the Western Atlantic. For example, in the order Gracilariales, we successfully assigned species names to most of the BINs, thanks to previous barcoding efforts in the region [58,87,91,94,95,101,102,103,104]. However, significant gaps remain in our knowledge of red macroalgae species in the Tropical Western Atlantic, particularly within the order Corallinales, where most barcoding efforts have been focused on non-geniculated species [31,32,35,36,60,61,62]. Despite some limited efforts to barcode geniculated species [105], critical data are still lacking to enable the identification of the newly discovered species diversity in Bocas del Toro.
Despite the high number of BINs identified in this study and the species previously characterized using DNA sequences (Table S5), the recorded diversity remains lower than the total number of species reported for the Caribbean coast of Panamá [42]. DNA barcoding efforts for red macroalgae in the Caribbean documented 126 species from 272 specimens. This is significantly fewer than the 227 species identified morphologically from approximately 2395 specimens in the Panamanian Caribbean [42].
This discrepancy reflects the longer history and widespread use of morphological methods and also highlights a key challenge in assessing species diversity using DNA barcoding: generating sequences can be more costly and time-intensive than traditional morphological identification. Consequently, there are currently more species reports through morphological assessments than through sequencing efforts. But although the number of specimens analyzed by morphology is much greater than that analyzed by sequencing, we have found that molecular analyses of DNA sequences can lead to the discovery of greater species richness.
A notable case is the genus Amphiroa. Morphological identification has recognized only six species and one variety in the Panamanian Caribbean (A. beauvoisii, A. fragilissima, A. hancockii, A. misakiensis, A. rigida, A. rigida var. antillana, and A. tribulus) [42]. However, our study identified 13 BINs within this genus (Table 1), suggesting that diversity in Amphiroa is underestimated when relying solely on morphological traits. While cox1 barcodes are available for three of these species (A. beauvoisii, A. fragilissima, and A. rigida) from various regions worldwide [105,106,107,108], only one BIN matched the cox1 from A. fragilissima and A. rigida [105]. This finding underscores the limited availability of genetic reference data and the potential for undiscovered species within the genus.
Morphological identifications in our dataset also revealed significant cryptic diversity. For example, samples identified morphologically as A. hancockii corresponded to three distinct BINs (ABA1815, ACW4703, and ACW4606), indicating the presence of multiple genetically distinct lineages within what has traditionally been recognized as a single species. This highlights the limitations of relying solely on morphology for species identification and emphasizes the importance of integrating molecular data to resolve hidden diversity.
In this study, we generated a substantial number of DNA sequences of red algae, but we did not identify any new amphi-isthmian species or samples found on either coast. This is a somewhat surprising outcome because the recent overall expansion of the Panamá Canal and salinity increases in some areas of Gatun lake [109] might have lessened the freshwater boundary for marine species to pass through this major waterway. Based on the current data gathered from GenBank and this study, the only known amphi-isthmian species remain those discovered in the last two decades from the family Rhodomelaceae [28,100,110] and the Ceramiaceae genus Centroceras [39]. More studies need to be performed to understand why only these small filamentous algae are found on both coasts, but not the fleshy and calcareous algae. Similarly, no newly introduced species were found in the newly generated sequences from the Bocas del Toro Archipelago. The only records of introduced species continue to come from Kappaphycus alvarezii samples sequenced and reported more than ten years ago [111,112,113]. In addition to this mariculture species, a few other DNA sequence-verified species are far from their type localities, suggesting that they might be introduced species in Panamá (Table S5). However, these names have been mainly applied based on similarities to the type specimens’ morphological descriptions, and the number of available type sequences is currently very low. With the continued expansion of type specimen sequencing, these potentially introduced species might be reassessed in the future. Additionally, it is important to note that the majority of species found in Bocas del Toro are native and have been reported from the Caribbean and Brazil.
Records based on the morphological identification of red algae in Panamá have documented a total of 21 orders [42]. While DNA barcoding efforts in Panamá over the last two decades have resulted in a significant number of sequences (Table S5), current sequences represent only 15 orders. No DNA barcodes have been generated for samples from the orders Acrosymphytales, Hildenbrandiales, Sebdeniales, or Stylonematales. Plocamiales, Rhodogorgonales, and Nemastomatales are represented by DNA sequences for only one specimen each. Future research in Panamá should prioritize collecting and barcoding samples from these orders to better document their molecular diversity, and special attention must be given to their ecological aspects to assess their conservation status.
Besides the lack of DNA sequences from several orders in Panamá, we also found that several regions on both coasts of Panamá are undersampled (Table S5). In the Caribbean, most sequences have been generated from the Bocas del Toro Archipelago and the area near the Galeta station of the Smithsonian Tropical Research Institute (STRI), in Colón Province. However, several areas remain poorly represented, such as the western part of Colón Province, the northern coast of Veraguas Province, and the entire Guna Yala region, which currently has only one sequence for the entire province. Similarly, on the Pacific coast, most samples have been collected from areas near the STRI Naos Marine Laboratory (Bay of Panamá) and the Burica Peninsula and some islands in the Gulf of Chiriquí (Table S5). However, the mainland coasts of the Gulf of Chiriquí and the Azuero Peninsula, as well as much of the Gulf of Panamá and the Pacific coast of Darién, remain underrepresented in DNA barcode data.

5. Conclusions

This study provides the largest DNA barcoding inventory of red algae from the Bocas del Toro Archipelago and the Caribbean coast of Panamá using COI-5P. A total of 179 sequences were obtained, clustering into 82 Barcode Index Numbers (BINs), with 16 representing new species records for the Caribbean coast of Panamá. More than half of the BINs lacked close matches in GenBank, indicating the possible presence of undescribed species. Phylogenetic analyses confirmed two new species, Gracilaria bocatorensis sp. nov. and Gracilaria dreckmannii sp. nov., supported by rbcL and cox1 sequences. Species delimitation analyses resolved taxonomic inconsistencies in Gracilaria and found that several samples identified as G. cervicornis and G. ferox are the same species, as well as the majority of the DNA sequences of samples identified as G. mammillaris, G. cuneata, and G. curtissiae. This study expands the knowledge of red algal diversity in the Caribbean and highlights the importance of DNA barcoding in uncovering cryptic diversity.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/d17040222/s1: Table S1: Pairwise rbcL p-distance comparisons between G. dreckmannii sp. nov. and other Gracilaria species within the G. mammillaris and G. cervicornis clade; Table S2: Pairwise cox1 p-distance comparisons between the two new Gracilaria species from Bocas del Toro and other Gracilaria species within their respective clades; Table S3: Pairwise UPA p-distance comparisons between the two new Gracilaria species from Bocas del Toro and other Gracilaria species within their respective clades; Table S4: Pairwise rbcL p-distance comparisons between G. bocatorensis sp. nov. and other Gracilaria species within the G. parva clade; Table S5: DNA barcodes of red marine macroalgae species (Rhodophyta) from the Caribbean and Pacific coast of Panamá published on GenBank; Supplement S6: Spanish summary. References [28,29,30,31,32,33,34,35,36,37,38,39,57,58,59,60,61,62,91,92,100,110,111,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128] are cited in the supplementary materials.

Author Contributions

Conceptualization, M.E.M.C., A.C.D., R.C., and D.W.F.; methodology, M.E.M.C. and A.C.D.; software, M.E.M.C.; validation, M.E.M.C. and D.W.F.; formal analysis, M.E.M.C.; investigation, M.E.M.C., S.F., B.W., D.W.F., A.C.D., and K.S.M.III; resources, D.W.F., A.C.D., and K.S.M.III; data curation, M.E.M.C., D.W.F., A.C.D., and K.S.M.III; writing—original draft preparation, M.E.M.C. and D.W.F.; writing—review and editing, all authors; visualization, M.E.M.C.; supervision, D.W.F.; project administration, S.F., B.W., D.W.F., A.C.D., and R.C.; funding acquisition, S.F., B.W., R.C., and D.W.F. All authors have read and agreed to the published version of the manuscript.

Funding

Funding for this project was provided by NSF OISE-0819205, PASI: Advanced tropical phycology: integrating modern and traditional techniques to the study of tropical algae to R. Collin, B. Wysor, and S. Fredericq and by the Smithsonian Institution MSN Workshop: Taxonomy, phylogeny, and DNA barcoding of Bocas del Toro seaweeds to Collin R., B. Wysor, W. Freshwater, S. Fredericq, J. Norris, and L. Weigt. All or portions of the laboratory and/or data analysis were conducted in and with the support of the Laboratories of Analytical Biology of the National Museum of Natural History. Additional financial support was provided by the Marine Science Network, the Center for Marine Science (UNCW) DNA—Algal Research Fund and the Red Pond Trust.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

All DNA sequences used in this study are in the BOLD project (https://dx.doi.org/10.5883/DS-RHOBOCAS) and can also be accessed in GenBank under accession numbers PV136053 to PV136231 and PV136446 to PV136450.

Acknowledgments

The authors would like to acknowledge the Smithsonian Tropical Research Institute and the staff at the Bocas del Toro Research Station for their invaluable assistance with the logistics of the Training in Tropical Taxonomy workshop. We are also grateful to the Laboratories of Analytical Biology, National Museum of Natural History, Smithsonian Institution in Washington for their support with sequencing samples.

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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Figure 1. Map of collection sites in the Bocas del Toro Archipelago: (1) Bastimento–Solarte Channel, (2) east side of Isla Carenero, (3) east of Wild Cane Cay, (4) Wild Cane Cay, (5) Bocas del Toro Archipelago (exact site unknown), (6) Bayside of STRI Bocas Laboratory, (7) Playa Istmito, (8) Flat Rock Beach, (9) Long Bay point two, (10) Long Bay point four, (11) Playa Boca Drago, (12) Tervi Bight, (13) Mimbi Timbi, and (14) Swan Cay.
Figure 1. Map of collection sites in the Bocas del Toro Archipelago: (1) Bastimento–Solarte Channel, (2) east side of Isla Carenero, (3) east of Wild Cane Cay, (4) Wild Cane Cay, (5) Bocas del Toro Archipelago (exact site unknown), (6) Bayside of STRI Bocas Laboratory, (7) Playa Istmito, (8) Flat Rock Beach, (9) Long Bay point two, (10) Long Bay point four, (11) Playa Boca Drago, (12) Tervi Bight, (13) Mimbi Timbi, and (14) Swan Cay.
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Figure 2. UPGMA cluster diagram based on 179 COI-5P sequences from Bocas del Toro, Panamá. Pairwise distances were calculated using raw p-distances. Branch tip labels display the BOLD BIN code, order, species, and number of duplicate sequences in between parenthesis. The tree scale represents the proportion of nucleotide differences calculated using the p-distance model. Node labels indicate the branch cluster support above 70% from 10,000 bootstrap replicates.
Figure 2. UPGMA cluster diagram based on 179 COI-5P sequences from Bocas del Toro, Panamá. Pairwise distances were calculated using raw p-distances. Branch tip labels display the BOLD BIN code, order, species, and number of duplicate sequences in between parenthesis. The tree scale represents the proportion of nucleotide differences calculated using the p-distance model. Node labels indicate the branch cluster support above 70% from 10,000 bootstrap replicates.
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Diversity 17 00222 g003
Figure 3. Histogram of DNA distance pairwise comparisons from sequences of Bocas del Toro specimens. Pairwise distances were calculated using the raw p-distances. Dashed black lines represent the COI-5P barcoding gap (1.13–2.71%).
Figure 3. Histogram of DNA distance pairwise comparisons from sequences of Bocas del Toro specimens. Pairwise distances were calculated using the raw p-distances. Dashed black lines represent the COI-5P barcoding gap (1.13–2.71%).
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Figure 4. Histogram of BLAST percentage identity for the 179 COI-5P sequences from Bocas del Toro. The dashed black line represents the minimum interspecific p-distance (2.71%) identified in the barcode gap analysis (Figure 3).
Figure 4. Histogram of BLAST percentage identity for the 179 COI-5P sequences from Bocas del Toro. The dashed black line represents the minimum interspecific p-distance (2.71%) identified in the barcode gap analysis (Figure 3).
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Diversity 17 00222 g005
Figure 5. Maximum likelihood phylogeny of Gracilariaceae based on rbcL (1467 bp). Node labels display bootstrap (BS) support and posterior probability (PP) for BS > 70% PP > 0.90. The scale bar represents substitutions per site, with the root (Gracilariopsis tenuifrons, GenBank accession MZ336075) removed for clarity. Tip labels include genus abbreviations, G. (Gracilaria), C. (Crassiphycus), A. (Agarophyton), H. (Hydropuntia), and Gp. (Gracilariopsis), species, locality, and GenBank accession or BOLD process ID. Bolded tips indicate sequences from Bocas del Toro, and numbers in parenthesis represent identical Bocas del Toro sequences excluded from the analysis.
Figure 5. Maximum likelihood phylogeny of Gracilariaceae based on rbcL (1467 bp). Node labels display bootstrap (BS) support and posterior probability (PP) for BS > 70% PP > 0.90. The scale bar represents substitutions per site, with the root (Gracilariopsis tenuifrons, GenBank accession MZ336075) removed for clarity. Tip labels include genus abbreviations, G. (Gracilaria), C. (Crassiphycus), A. (Agarophyton), H. (Hydropuntia), and Gp. (Gracilariopsis), species, locality, and GenBank accession or BOLD process ID. Bolded tips indicate sequences from Bocas del Toro, and numbers in parenthesis represent identical Bocas del Toro sequences excluded from the analysis.
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Diversity 17 00222 g006
Figure 6. Maximum likelihood phylogeny of Gracilariaceae based on cox1 (1626 bp). The tree description is similar to that in Figure 5. The tree root (Melanthalia obtusata, GenBank accession MH396016) was removed for clarity.
Figure 6. Maximum likelihood phylogeny of Gracilariaceae based on cox1 (1626 bp). The tree description is similar to that in Figure 5. The tree root (Melanthalia obtusata, GenBank accession MH396016) was removed for clarity.
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Diversity 17 00222 g007
Figure 7. Maximum likelihood phylogenetic reconstruction of a concatenated dataset of rbcL + UPA + cox1 (3451 bp) with species delimitation analyses. Leaf labels denote species, voucher, country, and GenBank accession numbers. Bold labels indicate sequences obtained in this study. The tree scale represents the number of substitutions per site. All the bootstrap and posterior probability values are shown at the nodes. Gray boxes represent the results of the ABGD, while the dashed boxes represent the results of the mPTP species delimitation. Outgroup (Gracilaria flabelliformis subsp. Simplex, GenBank accessions MZ336059 and MZ336088) was removed from the tree to aid visualization.
Figure 7. Maximum likelihood phylogenetic reconstruction of a concatenated dataset of rbcL + UPA + cox1 (3451 bp) with species delimitation analyses. Leaf labels denote species, voucher, country, and GenBank accession numbers. Bold labels indicate sequences obtained in this study. The tree scale represents the number of substitutions per site. All the bootstrap and posterior probability values are shown at the nodes. Gray boxes represent the results of the ABGD, while the dashed boxes represent the results of the mPTP species delimitation. Outgroup (Gracilaria flabelliformis subsp. Simplex, GenBank accessions MZ336059 and MZ336088) was removed from the tree to aid visualization.
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Diversity 17 00222 g008
Figure 8. Maximum likelihood phylogeny of Gracilaria based on rbcL (1467 bp). This phylogeny includes all publicly available sequences from species within the Gracilaria mammillaris and Gracilaria cervicornis clades. Species names in parentheses indicate previous identifications in GenBank or those determined by Lyra et al. [87]. Red tip labels indicate sequences that belong to one species in the rbcL phylogeny but are part of a different species in the cox1 or UPA phylogeny. Further tree labeling characteristics are as described for Figure 7.
Figure 8. Maximum likelihood phylogeny of Gracilaria based on rbcL (1467 bp). This phylogeny includes all publicly available sequences from species within the Gracilaria mammillaris and Gracilaria cervicornis clades. Species names in parentheses indicate previous identifications in GenBank or those determined by Lyra et al. [87]. Red tip labels indicate sequences that belong to one species in the rbcL phylogeny but are part of a different species in the cox1 or UPA phylogeny. Further tree labeling characteristics are as described for Figure 7.
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Diversity 17 00222 g009
Figure 9. Maximum likelihood phylogeny of Gracilaria based on cox1 (1614 bp). Species names in parentheses indicate previous identifications in GenBank or those determined by Lyra et al. [87]. Further tree labeling characteristics are as described for Figure 7.
Figure 9. Maximum likelihood phylogeny of Gracilaria based on cox1 (1614 bp). Species names in parentheses indicate previous identifications in GenBank or those determined by Lyra et al. [87]. Further tree labeling characteristics are as described for Figure 7.
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Diversity 17 00222 g010
Figure 10. Maximum likelihood phylogeny of Gracilaria based on UPA (370 bp). Species names in parentheses indicate previous identifications in GenBank or those determined by Lyra et al. [87]. Further tree labeling characteristics are as described for Figure 7.
Figure 10. Maximum likelihood phylogeny of Gracilaria based on UPA (370 bp). Species names in parentheses indicate previous identifications in GenBank or those determined by Lyra et al. [87]. Further tree labeling characteristics are as described for Figure 7.
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Diversity 17 00222 g011
Figure 11. Gracilaria bocatorensis sp. nov. habit and internal morphology. (a) Female thallus habit with mature cystocarps. (b) Vegetative thallus habit. (c,d) Cross-section of the mid-section of the blade. (e) Cross-section view of the cortical cells. Figures taken from specimens: WNC34630, field ID collection number TFP08-229 (PHYKOS-5359) (a,c), WNC34521, field ID collection number TFP08-233 (PHYKOS-5363) holotype (b,d,e).
Figure 11. Gracilaria bocatorensis sp. nov. habit and internal morphology. (a) Female thallus habit with mature cystocarps. (b) Vegetative thallus habit. (c,d) Cross-section of the mid-section of the blade. (e) Cross-section view of the cortical cells. Figures taken from specimens: WNC34630, field ID collection number TFP08-229 (PHYKOS-5359) (a,c), WNC34521, field ID collection number TFP08-233 (PHYKOS-5363) holotype (b,d,e).
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Diversity 17 00222 g012
Figure 12. Internal morphology of the cystocarp in Gracilaria bocatorensis sp. nov. WNC34630, field ID collection number TFP08-229 (PHYKOS-5359). (a) Cross-section of an immature, developing cystocarp. (b) Magnified cross-section of the cystocarp floor region, showing the inner pericarp (white bracket) and tubular nutritive cells (arrows). (c) Cross-section of the outer pericarp. (d) Cross-section view of a mature cystocarp. (e) Mature carposporangia (arrows).
Figure 12. Internal morphology of the cystocarp in Gracilaria bocatorensis sp. nov. WNC34630, field ID collection number TFP08-229 (PHYKOS-5359). (a) Cross-section of an immature, developing cystocarp. (b) Magnified cross-section of the cystocarp floor region, showing the inner pericarp (white bracket) and tubular nutritive cells (arrows). (c) Cross-section of the outer pericarp. (d) Cross-section view of a mature cystocarp. (e) Mature carposporangia (arrows).
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Diversity 17 00222 g013
Figure 13. Gracilaria dreckmannii sp. nov. habit and internal morphology. (a,b) Habit of rehydrated specimens. (c) Dried herbarium sheet specimen. (d,e) Cross-section of the upper part of the thalli. (f,g) Cross-section view of the cortical cells. (h) Surface view of the cortical cells. Figures taken from specimens: PHYKOS-5449 holotype (a,d,e,h), PHYKOS-5454 (b,f,g), and Madrid M. 472 (c).
Figure 13. Gracilaria dreckmannii sp. nov. habit and internal morphology. (a,b) Habit of rehydrated specimens. (c) Dried herbarium sheet specimen. (d,e) Cross-section of the upper part of the thalli. (f,g) Cross-section view of the cortical cells. (h) Surface view of the cortical cells. Figures taken from specimens: PHYKOS-5449 holotype (a,d,e,h), PHYKOS-5454 (b,f,g), and Madrid M. 472 (c).
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Figure 14. Internal morphology of the cystocarp in Gracilaria dreckmannii sp. nov. PHYKOS-5454. (a,b) Magnified cross-section of the cystocarp floor region (white bracket). (c) Cross-section of the outer pericarp. (d) Mature carposporangia (arrows). (e) Tubular nutritive cells (arrows) connecting the inner gonimoblast and the outer pericarp.
Figure 14. Internal morphology of the cystocarp in Gracilaria dreckmannii sp. nov. PHYKOS-5454. (a,b) Magnified cross-section of the cystocarp floor region (white bracket). (c) Cross-section of the outer pericarp. (d) Mature carposporangia (arrows). (e) Tubular nutritive cells (arrows) connecting the inner gonimoblast and the outer pericarp.
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Table 1. Samples sequenced in this study and the results of the COI-5P NCBI BLAST search. 1 First report from Caribbean Panamá; 2 COI-5P sequences from operational taxonomic units not represented in GenBank or BOLD SYSTEMS; 3 rbcL sequence; 4 UPA sequence. BOLD process ID, field ID, and voucher and collection site information can be found in the BOLD project (https://dx.doi.org/10.5883/DS-RHOBOCAS).
Table 1. Samples sequenced in this study and the results of the COI-5P NCBI BLAST search. 1 First report from Caribbean Panamá; 2 COI-5P sequences from operational taxonomic units not represented in GenBank or BOLD SYSTEMS; 3 rbcL sequence; 4 UPA sequence. BOLD process ID, field ID, and voucher and collection site information can be found in the BOLD project (https://dx.doi.org/10.5883/DS-RHOBOCAS).
Taxon and BOLD BINMaximum NCBI BLAST % Identity and GenBank AccessionsGenBank Accessions
CORALLINALES
2 Corallinales ACW461289.2% Corallinaceae HQ544204PV136126
2 Corallinales ADB537390.8% Titanoderma sp. OQ943746PV136176
2 Corallinales ADC039187.7% Cheilosporum cultratum (Harvey) Areschoug 1852 LC071762PV136224
Amphiroa cf. fragilissima (Linnaeus) J.V.Lamouroux 1816
AAO5848		99.6% Amphiroa cf. fragilissima (Linnaeus) J.V.Lamouroux 1816 MG521326PV136085
PV136212
PV136230
2 Amphiroa hancockii W.R.Taylor 1942 ABA181596.2–96.6% Corallinales GQ917680PV136132
PV136054
PV136104
PV136092
PV136077
PV136089
PV136070
PV136141
PV136180
PV136155
PV136137
PV136120
Amphiroa rigida J.V.Lamouroux 1816 ACL223598.2% Amphiroa rigida J.V.Lamouroux 1816 MG521335PV136170
2 Amphiroa sp. AEC741595.2% Lithophyllum corallinae (P.Crouan & H.Crouan) Heydrich 1897 MG521354PV136164
2 Amphiroa sp. AEC747896.7% Lithophyllum corallinae (P.Crouan & H.Crouan) Heydrich 1897 MG521354PV136098
2 Amphiroa sp. ACW305591.1% Amphiroa beauvoisii J.V.Lamouroux 1816 LC071729PV136066
PV136106
PV136172
2 Amphiroa sp. ACW419591.9% Amphiroa sp. SA-2023 LC767501PV136067
PV136096
PV136078
2 Amphiroa sp. ACW437593.7–94.1% Amphiroa beauvoisii J.V.Lamouroux 1816 LC071729PV136133
PV136109
PV136185
PV136118
2 Amphiroa sp. ACW453795.3% Corallinales GQ917303PV136163
PV136149
Amphiroa sp. ACW460698.2–98.3% Corallinales GQ917679PV136211
PV136158
PV136139
PV136058
PV136123
PV136099
PV136074
PV136196
PV136084
PV136201
PV136143
2 Amphiroa sp. ACW470392.8–93.1% Amphiroa foliacea J.V.Lamouroux 1824 OM460663PV136129
PV136203
Amphiroa sp. ADB556999.8% Corallinales GQ917676PV136081
2 Amphiroa sp. ADB628792.9% Corallinales GQ917652PV136151
1 Jania pedunculata var. adhaerens (J.V.Lamouroux)
A.S.Harvey,
Woelkerling & Reviers 2020 ACW3073		99.2% Jania pedunculata var. adhaerens (J.V.Lamouroux) A.S.Harvey, Woelkerling &
Reviers 2020 OR192975		PV136090
1,2 Jania sp. ADB566297–97.3% Jania rosea (Lamarck) Decaisne 1842 LC071777PV136204
PV136193
GRACILARIALES
1 Gracilaria baiana Lyra, Gurgel, M.C.Oliveira & Nunes 2016 ACP629598.8% Gracilaria baiana Lyra, Gurgel, M.C.Oliveira & Nunes 2016 KP210196PV136114
PV136202
PV136117
PV136227
PV136194
PV136207
1 Crassiphycus caudatus (J.Agardh) Gurgel, J.N.Norris &
Fredericq 2018 ACW4841		97.6–99.5% Gracilaria caudata (J.Agardh) Gurgel, J.N.Norris & Fredericq 2018 KP210151PV136072
PV136186
PV136095
PV136157
PV136184
PV136182
PV136144
1,2 Gracilaria bocatorensis sp. nov. ADB688595.2% Gracilaria sp. KY656553PV136199
3 PV136446
4 PV136450
PV136111
Gracilaria cearensis (A.B.Joly & Pinheiro) A.B.Joly & Pinheiro
1966 ABX1742		99.8% Gracilaria cearensis (A.B.Joly & Pinheiro) A.B.Joly & Pinheiro 1966
KP210189		PV136135
1 Crassiphycus corneus (J.Agardh) Gurgel, J.N.Norris & Fredericq 2018 AAV695399.5–99.7% Hydropuntia cornea (J.Agardh) M.J.Wynne 1989 KP636771PV136055
PV136116
PV136225
Crassiphycus crassissimus (J.Agardh) Gurgel, J.N.Norris &
Fredericq 2018 ACW3661		99.4–99.8% Crassiphycus crassissimus (J.Agardh) Gurgel, J.N.Norris & Fredericq 2018 MZ336084PV136190
PV136206
PV136173
1,2 Gracilaria dreckmannii sp. nov. ACW430095.6% Gracilaria microcarpa Dreckmann, Núñez-Resendiz & Sentíes 2018 MF321896PV136121
3 PV136444
4 PV136448
PV136071
3 PV136443
4 PV136447
1 Gracilaria flabelliformis subsp. simplex Gurgel, Fredericq & J.N.Norris
2004 ACP5692		99.5–99.8% Gracilaria flabelliformis subsp. simplex Gurgel, Fredericq & J.N.Norris 2004 KP210178PV136119
PV136102
PV136112
PV136076
PV136226
PV136087
PV136195
Gracilaria hayi Gurgel, Fredericq & J.N.Norris 2004 ABX1121100% Gracilaria hayi Gurgel, Fredericq & J.N.Norris 2004 KP210179PV136103
Gracilaria mammillaris (Montagne) M.Howe 1918 ABX112298.6–98.8% Gracilaria mammillaris (Montagne) M.Howe 1918 KP210166PV136115
PV136083
PV136189
1 Gracilaria microcarpa Dreckmann, Núñez-Resendiz &
Sentíes 2018 ACW4301		99.1% Gracilaria microcarpa Dreckmann, Núñez-Resendiz & Sentíes 2018
MF321896		PV136192
PV136222
PV136079
PV136154
PV136181
Gracilaria silviae Lyra, Gurgel, M.C.Oliveira &
J.M.C.Nunes 2015 ABX1150		99.8% Gracilaria silviae Lyra, Gurgel, M.C.Oliveira & J.M.C.Nunes 2015
KP210168		PV136062
1 Gracilaria suzanneae L.P.Soares, Gurgel & M.T.Fujii 2018
AGE7357		97.4% Gracilaria occidentalis (Børgesen) M.Bodard 1965 MW924175PV136165
3 PV136445
4 PV136449
1 Gracilariopsis silvana Gurgel, Fredericq & J.N.Norris 2003
ABX1323		99.8% Gracilariopsis silvana Gurgel, Fredericq & J.N.Norris 2003 KP210202PV136053
PV136205
PV136231
PV136191
PV136215
PV136145
PV136093
PV136105
CERAMIALES
2 Ceramiales AAV886385.7–85.8% Membranoptera alata (Hudson) Stackhouse 1809 JX111884PV136061
PV136229
PV136124
PV136177
PV136213
PV136169
2 Ceramiales ACW345190.5% Acanthophora dendroides Harvey 1855 MT876665PV136187
2 Ceramiales ACW477786.7% Placophora monocarpa (Montagne) Papenfuss 1956 KU564367PV136136
Acanthophora spicifera (Vahl) Børgesen 1910 AAF1313100% Acanthophora spicifera (Vahl) Børgesen 1910 MH388705PV136069
PV136064
PV136128
2 Aglaothamnion sp. ACW431888.8% Callithamnion acutum Kylin 1925 PP735967PV136088
Alsidium triquetrum (S.G.Gmelin) Trevisan 1845 ACW470599.7% Alsidium triquetrum (S.G.Gmelin) Trevisan 1845 MN165082PV136130
PV136065
PV136073
PV136056
PV136125
2 Augophyllum sp. ACW372695.2% Augophyllum wysorii Showe M.Lin, Fredericq & Hommersand 2004 OM460639PV136161
Centroceras gasparrinii (Meneghini) Kützing 1849 ADB685398.2% Centroceras gasparrinii (Meneghini) Kützing 1849 KP222749PV136228
2 Chondrophycus sp. AGE993293.6–93.8% Chondrophycus sp. MZ855311PV136167
PV136059
2 Corallophila sp. AGG135897.4% Corallophila sp. MW354762PV136214
2 Laurencia sp. AAO544096.6% Laurencia dendroidea J.Agardh 1852 MH388711PV136159
2 Laurencia sp. ACW366795% Laurencia australis M.Preuss, Diaz-Tapia, Verbruggen & Zuccarello 2023 OQ863292PV136188
PV136091
2 Laurencia sp. AEC744796.3% Laurencia dendroidea J.Agardh 1852 MH388711PV136122
1 Laurencia catarinensis Cordeiro-Marino & Fujii 1985
AAO5439		99.8% Laurencia catarinensis Cordeiro-Marino & Fujii 1985 OQ863298PV136097
Melanothamnus pseudovillum (Hollenberg) Díaz-Tapia
& Maggs 2017 AGE9893		99.8% Melanothamnus pseudovillum (Hollenberg) Díaz-Tapia & Maggs 2017 HM573524PV136140
2 Palisada sp. ACW391191.4% Palisada corallopsis (Montagne) Sentíes, M. T. Fujii & Díaz-Larrea 2008 PP974343PV136221
PV136152
2 Palisada sp. ADC048096.97–96.98% Palisada perforata (Bory) K.W.Nam 2007 MH388710PV136068
PV136209
2 Spyridia sp. AGE977797.4% Spyridia americana Durant 1850 MW770747PV136219
1 Vidalia obtusiloba (Mertens ex C.Agardh) J.Agardh 1863
ACW3810		99.5% Vidalia obtusiloba (Mertens ex C.Agardh) J.Agardh 1863 MG188846PV136138
Wrangelia argus (Montagne) Montagne 1856 AAO9061100% Wrangelia argus (Montagne) Montagne 1856 OQ561846PV136086
2 Wrangelia sp. ACW368993.4% Wrangelia ryancraigii C.W.Schneider & G.W.Saunders 2024 OQ561822PV136110
GIGARTINALES
1 Hypnea cryptica P.B.Jesus & J.M.C.Nunes 2019 AEC692099.5% Hypnea cryptica P.B.Jesus & J.M.C.Nunes 2019 OR589246PV136217
1 Hypnea pseudomusciformis Nauer, Cassano & M.C.Oliveira
2015 AGH7665		99.35–99.57% Hypnea pseudomusciformis Nauer, Cassano & M.C.Oliveira 2015 MH482171PV136153
PV136107
PV136174
2 Hypnea sp. AEC724797.4% Hypnea musciformis (Wulfen) J.V.Lamouroux 1813 PP898380PV136179
1 Hypnea wynnei NOBIN2100% Hypnea wynnei Nauer, Cassano & M.C.Oliveira 2016 MZ855294PV136162
Solieria sp. ACW472398.5% Solieria sp. MG018946PV136220
PV136210
PV136156
PV136200
2 Solieria sp. AGG346697.3% Solieria filiformis (Kützing) P.W.Gabrielson 1985 KJ202080PV136168
PV136108
2 Solieria sp. AGE978293.4% Solieria sp. MG018946PV136075
RHODYMENIALES
2 Rhodymeniales ACW373090.6% Lomentaria sp. KU707864PV136094
PV136147
2 Rhodymeniales AED078788.6–89% Neogastroclonium subarticulatum (Turner) L.Le Gall, Dalen &
G.W.Saunders 2008 MN447949		PV136178
PV136100
2 Botryocladia sp. AEC742294% Botryocladia skottsbergii (Børgesen) Levring 1941 HQ423132PV136080
Botryocladia sp. AEC797198.8% Botryocladia sp. KR011965PV136223
2 Botryocladia sp. AED068191% Botryocladia leptopoda (J.Agardh) Kylin 1931 MT876667PV136148
Ceratodictyon intricatum (C.Agardh) R.E.Norris 1987
AAO6470		99.8–100% Ceratodictyon intricatum (C.Agardh) R.E.Norris 1987 OK641564PV136160
PV136218
NEMALIALES
Dichotomaria sp. ACW348599.3–99.7% Dichotomaria sp. KF752542PV136197
PV136175
PV136057
PV136131
2 Galaxaura sp. ADB548396.2% Galaxaura rugosa (J.Ellis & Solander) J.V.Lamouroux 1816 MT472792PV136060
Galaxaura rugosa (J.Ellis & Solander) J.V.Lamouroux 1816
AAO6697		99.4% Galaxaura rugosa (J.Ellis & Solander) J.V.Lamouroux 1816 MT472804PV136134
2 Scinaia sp. ACW457695.5% Scinaia hormoides Setchell 1914 HQ422966PV136082
2 Tricleocarpa sp. ACW382996.7% Tricleocarpa cylindrica (J.Ellis & Solander) Huisman & Borowitzka 1990 KY656534PV136198
Tricleocarpa fragilis (Linnaeus) Huisman & R.A.Townsend
1993 AAO8732		99.4% Tricleocarpa fragilis (Linnaeus) Huisman & R.A.Townsend 1993 KU321670PV136150
GELIDIALES
1 Gelidiella flabella G.H.Boo & Le Gall 2016 NOBIN199.8% Gelidiella flabella G.H.Boo & Le Gall 2016 KT207971PV136063
Gelidium lineare Iha & Freshwater 2016 AAU034698.9% Gelidium lineare Iha & Freshwater 2016 KT208015PV136183
2 Gelidium sp. AAO643297.9% Gelidium microdonticum W.R.Taylor 1969 KT208002PV136166
HALYMENIALES
Corynomorpha clavata (Harvey) J.Agardh 1872 ACJ252498.9% Corynomorpha clavata (Harvey) J.Agardh 1872 MK919031PV136127
Grateloupia sp. ACR934999.7% Grateloupia sp. MK919065PV136113
2 Halymenia sp. AEC904393.3% Halymenia durvillei Bory 1828 MK812802PV136101
PEYSSONNELIALES
2 Peyssonneliales AEC899393.2% Peyssonneliales sp. 1 OM902126PV136208
2 Peyssonnelia sp. ACW431592% Peyssonnelia sp. HQ545338PV136146
2 Peyssonnelia sp. ADB654091.9% Peyssonnelia sp. JX969736PV136142
PV136171
2 Polystrata sp. AGE991593.4% Polystrata sp. OM902137PV136216
Table 2. Intraspecific and interspecific pairwise genetic distances among the new species within the clade of Gracilaria parva and Gracilaria cervicornis. Pairwise genetic distances were calculated using p-distances. Detailed pairwise comparisons for all samples are provided in Supplementary Tables S1–S4.
Table 2. Intraspecific and interspecific pairwise genetic distances among the new species within the clade of Gracilaria parva and Gracilaria cervicornis. Pairwise genetic distances were calculated using p-distances. Detailed pairwise comparisons for all samples are provided in Supplementary Tables S1–S4.
LociSpeciesMean
Intra. (%)		Maximum
Intra. (%)		Nearest NeighborMin. Dist.
to NN (%)
rbcLG. apiculata0.390.76G. cervicornis1.84
G. cervicornis (G. ferox)0.360.89G. apiculata1.84
G. dreckmannii sp. nov.00G. microcarpa1.58
G. mammillaris (G. curtissiae and G. cuneata)0.651.12Gracilaria sp. SPF571662.80
G. microcarpa0.501.41G. dreckmannii sp. nov.1.58
Gracilaria sp. SPF5716600G. mammillaris2.80
cox1G. cervicornis (G. ferox)1.993.29G. dreckmannii sp. nov.4.52
G. dreckmannii sp. nov.00G. microcarpa4.43
G. mammillaris (G. curtissiae and G. cuneata)1.632.57G. cervicornis6.42
G. microcarpa1.011.47G. dreckmannii sp. nov.4.43
Gracilaria sp. SPF5716600G. dreckmannii sp. nov.5.57
UPAG. cervicornis (G. ferox)0.540.54Gracilaria sp. SPF571660.81
G. mammillaris (G. curtissiae and G. cuneata)0.40.54Gracilaria sp. SPF571661.08
G. dreckmannii sp. nov.00Gracilaria sp. SPF571661.08
Gracilaria sp. SPF5716600G. cervicornis0.81
Table 3. Morphological comparison between Gracilaria bocatorensis and other closely related species.
Table 3. Morphological comparison between Gracilaria bocatorensis and other closely related species.
CharactersG. bocatorensis
(This Study)		G. galetensis
[91]		G. parva
[92]		G. gurgelii
[91]		G. oliveirarum
[91]		G. smithsoniensis [91]G. hayi
[91]		G. occidentalis
[91,93]
ThallusCompressed to flattenedFlattenedCompressed to flattenedFlattenedFlattenedCompressedFlattenedFlattened
Plant length (cm)2.5–7.78.0–20.0Up to 2.57.0–10.010.0–14.03.0–5.55.0–15.0Up to 25
Axis width (mm)1.7–4.45.0–8.00.5–4.05.0–8.05.0–12.02.0–2.55.0–25.04.0
Thallus thickness (µm)328–417254–275-250–330500–750‘Thin”, up to 300‘Thin’, up to 100ca. 800 at margins
HoldfastNot observedSmall, discoidSmall, discoidSmall, tough,
irregular shaped		Small, discoidSmall, discoidDiscoid-
BranchingSubdichotomous to irregularDichotomous to subdichotomousDichotomous or cervicornDichotomous to subdichotomousDichotomous to subdichotomous to irregularDichotomousDichotomous to subdichotomousDichotomous to subdichotomous
Orders of branchingUp to 52–3Up to 42–3 (3+) 13–5+Up to 5+Up to 5+Up to 5+
ApicesObtuseObtuseObtuse to truncateObtuseNot describedObtuseObtuse-
Cortical cell layers1–31–32–31–21–21–21–21–2
Cortical cell
shape		QuadrateVariable, mostly anticlinally elongatedPolyhedralIsodiametric, sometimes anticlinally elongatedVariableVariable, periclinally to anticlinally elongatedPericlinally compressedRounded polyhedral, often compressed
Cortical cell
diameter (µm)		2.7–10.03.75–12.54.0–10.03.75–11.83.0–9.03.7–7.52.5–10.05.0–16.2
Medullary cell layers4–84–53–64–54–55–63–55–6
Medullary cell shapeGloboseRounded, isodiametric, most slightly compressedGlobose to longitudinally ovoidIsodiametric–slightly compressedInner cells ovoid, outer globose to ovoidIsodiametricGlobose to ovoidGlobose to ovoid
Medullary cell
diameter (µm)		56.0–102.056.0–154.070.0–160.0100.0–185.0Inner cells 150.0–400.061.7–116.050.0–86.0103.0–135.0
Cortex-to-medulla transitionAbruptGradualAbruptAbruptAbruptAbruptAbruptAbrupt
Spermatia typeNot observedShallow textorii-typeShallow textorii-type--Shallow textorii-typeShallow textorii-type-
Cystocarp shapeHemisphericalHemispherical to urceolateHemispherical--HemisphericalHemispherical-
Cystocarp diameter (mm)0.7–0.8Up to 1.0Not described--1.0–2.01.0–2.0-
Outer pericarp cell layers9–1112–1411–17--12–1412–14-
Tubular nutritive cellsFew, attached to inner pericarpNot describedAttached to inner and outer pericarp--Attached to lower outer pericarpProminent, attached to outer pericarp-
1 All specimens illustrated in Gurgel et al. [91] were damaged thalli.
Table 4. Morphological comparison between Gracilaria dreckmannii and other closely related species.
Table 4. Morphological comparison between Gracilaria dreckmannii and other closely related species.
CharactersG. dreckmannii (This Study)G. cervicornis
[87,88,94]		G. macrocarpa
[88]		G. mammillaris
[91]		G. cuneata
[94,95]		G. ferox
[87,88]		G. apiculate
[96]
ThallusCylindricalFlattenedCylindricalFlattenedFlattenedFlattened mostlyFlattened, occasionally cylindrical
Plant length (cm)2.4–4.86.0–35.08.0–22.0-3.0–12.0Up to 38.05.0–25.0
Axis width (mm)1.9–3.0-0.43–0.62-10.0–20.0To 2.02–4 (flattened); 1+ (cylindrical
Thallus thickness (µm)660–1200-430–620620–1000600–1000-Not described
HoldfastDiscoid-Small, discoid--Irregular, cushion-likeSmall, discoid
BranchingSubdichotomous to irregularSparse, irregularDichotomous sometimes trichotomous-Mostly dichotomous-Mostly alternate but variable
Orders of branchingUp to 7Up to 24–5+---Up to 3
Apices-AcuteAcute-Obtuse to acute-Not described
Cortical cell layers1–22–32–31–21–22–71–2
Cortical cell shapeGlobose to elongate-SphericalIsodiametricQuadraticRadially elongateRadially elongate, elipsoid
Cortical cell diameter (µm)3–105–92–73–77–10-3–11
Medullary cell layers7–114–68–93–42–52–56–12
Medullary cell shapeOvoid to globoseOvoid to polyhedralIsodiometric to irregularOvoid to globose--Rounded to compressed
Medullary cell diameter (µm)88–11755–13330–110261–39825–20712–1570–175
Cortex-to-medulla transitionAbruptGradualAbruptAbruptGradualGradualSomewhat gradual
Spermatia typeNot observedShallow textorii-typeNot observed-Shallow textorii-typeShallow textorii-typeNot observed
Cystocarp shapeDome shaped constricted around the baseSubspherical and pedunculateHemispherical to subspherical and pedunculate-Hemispherical or subspherical and basally constricted-Variable, some urceolate and pedunculate
Cystocarp diameter (mm)0.50.75–0.800.28–0.30--0.50–1.001.5–2.0
Outer pericarp cell layers9–116–1012–14-10–148–129–13
Tubular nutritive cellsAttached to outer pericarpAttached to chamber floorAttached to outer pericarp-Attached to chamber floor, rarely to outer pericarpAbsentAbundant, attached to outer pericarp and chamber floor
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MDPI and ACS Style

Madrid Concepcion, M.E.; Collin, R.; Macdonald, K.S., III; Driskell, A.C.; Fredericq, S.; Wysor, B.; Freshwater, D.W. DNA Barcoding of Red Algae from Bocas del Toro, Panamá, with a Description of Gracilaria bocatorensis sp. nov. and G. dreckmannii sp. nov. (Gracilariales, Gracilariaceae). Diversity 2025, 17, 222. https://doi.org/10.3390/d17040222

AMA Style

Madrid Concepcion ME, Collin R, Macdonald KS III, Driskell AC, Fredericq S, Wysor B, Freshwater DW. DNA Barcoding of Red Algae from Bocas del Toro, Panamá, with a Description of Gracilaria bocatorensis sp. nov. and G. dreckmannii sp. nov. (Gracilariales, Gracilariaceae). Diversity. 2025; 17(4):222. https://doi.org/10.3390/d17040222

Chicago/Turabian Style

Madrid Concepcion, Maycol Ezequiel, Rachel Collin, Kenneth S. Macdonald, III, Amy C. Driskell, Suzanne Fredericq, Brian Wysor, and D. Wilson Freshwater. 2025. "DNA Barcoding of Red Algae from Bocas del Toro, Panamá, with a Description of Gracilaria bocatorensis sp. nov. and G. dreckmannii sp. nov. (Gracilariales, Gracilariaceae)" Diversity 17, no. 4: 222. https://doi.org/10.3390/d17040222

APA Style

Madrid Concepcion, M. E., Collin, R., Macdonald, K. S., III, Driskell, A. C., Fredericq, S., Wysor, B., & Freshwater, D. W. (2025). DNA Barcoding of Red Algae from Bocas del Toro, Panamá, with a Description of Gracilaria bocatorensis sp. nov. and G. dreckmannii sp. nov. (Gracilariales, Gracilariaceae). Diversity, 17(4), 222. https://doi.org/10.3390/d17040222

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