Molecular data demonstrate the occurrence of Compsopogon caeruleus (Compsopogonophyceae, Rhodophyta) in Bahia state, BrazilAbstract
The freshwater red macroalga Compsopogon caeruleus is ubiquitously distributed in tropical and subtropical regions and has been reported in many Brazilian states. Here, we report the first record of this species in the state of Bahia, constituting the third record of this species in the northeast region. This population was found on an oyster farm in the Graciosa Quilombola Fishing Community, Taperoá, Bahia, Brazil. We conducted a detailed morphological assessment and analyzed of the sequences of the molecular markers COI-5P and rbcL.
Key words:
Brazilian northeast; COI-5P; Compsopogononales; rbcL; red algae
Resumo
Embora seja ubiquamente distribuída em regiões tropicais e subtropicais, e seja relatada para muitos estados brasileiros, a macroalga vermelha de água doce Compsopogon caeruleus é relatada pela primeira vez para o estado da Bahia, constituindo a terceira citação para a região Nordeste. A população foi encontrada em uma fazenda de ostras na Comunidade de Pesca Quilombola Graciosa, Taperoá, Bahia, Brasil. Neste estudo, descrevemos sua morfologia em detalhes, corroborando com análises das sequências dos marcadores moleculares COI-5P e rbcL.
Palavras-chave:
nordeste brasileiro; COI-5P; Compsopogononales; rbcL; algas vermelhas
Introduction
The freshwater macroalgae Compsopogon caeruleus (Balbis ex C.Agardh) Montagne (1846:154) (Compsopogonophyceae, Rhodophyta) is widely distributed in tropical and subtropical regions (Oliveira Filho & Pereira 1973; South & Skelton 2002; Liu & Wang 2004; Ratha et al. 2007; Sherwood et al. 2010; Kitayana 2011; Bautista & Necchi Junior 2014; Vis & Necchi Junior 2021). The species has even been reported in temperate regions (Nichols 1964; Chapman & Cameron 1967; Žáková et al. 2013; Zaburlín et al. 2019; Vis & Necchi Junior 2021), where it is often considered an invasive species (Žáková et al. 2013; Zaburlín et al. 2019).
Compsopogon caeruleus is well adapted to adverse environmental conditions, including aquariums and artificial lakes, natural water bodies affected by the discharge of industrial effluents (Szymanska & Krupinska 1983; Scott et al. 1988; Scott & Broadwater 1989; Stoyneva et al. 2006; Žáková et al. 2013; Bautista & Necchi Junior 2014), and water discharge regions that are used to cool power plants (Chapman & Cameron 1967).
Advancements in morphological taxonomic studies and, subsequently, the emergence of molecular taxonomy have resulted in the consolidation of most Compsopogon taxa, with 19 heterotypic synonyms now included in C. caeruleus (Guiry 2022). This has expanded its global distribution, demonstrating its remarkable phenotypic plasticity and adaptability. Molecular studies have revealed that although C. caeruleus populations are geographically isolated, they exhibit low genetic diversity (Necchi Junior et al. 2013; Nan et al. 2016; Zaburlín et al. 2019).
According to Flora e Funga do Brasil 2025 (continuously updated), C. caeruleus is documented from the north to the south of Brazil, in the states of Amazonas, Pará, Maranhão, Espírito Santo, Rio de Janeiro, São Paulo, Paraná, and Rio Grande do Sul; Necchi Junior et al. (2013) also mentioned its presence in the state of Sergipe. However, the species has not been confirmed in the state of Bahia. Here, we report the first documented occurrence of C. caeruleus in Bahia and provide morphological and molecular analyses.
Materials and Methods
Sample collection
A population of Compsopogon caeruleus was discovered growing in great abundance on an estuarine oyster farm in Graciosa, a community of quilombola fishermen (descendants of enslaved people), in the municipality of Taperoá, Bahia, Brazil (13°28′46.26″S, 39°05′34.50″W). The estuary is surrounded by mangroves, and the land is occupied by extensive palm oil plantations.
The highest occurrence of algae occurred in autumn 2022, the region’s rainiest period, when local oyster farmers recorded salinities ranging from approximately 8 at low tide to 24 at high tide. The algae were growing in the oyster farming lanterns in dense populations, harming cultivation because of the weight of the floating structures.
Sampling was conducted in July 2022, and the collected material was preserved in 70% alcohol. The material was subsequently used for morphological and molecular analyses. A voucher specimen was deposited in the Herbarium Alexandre Leal Costa (ALCB) at the Federal University of Bahia, Brazil (Tab. 1).
Species, collection information, and voucher and accession numbers of COI-5P and rbcL sequences.
Morphological analysis
Aspects of external and internal morphology were analyzed using a stereomicroscope MoticTM-SMZ-168 Series (Hong Kong, China), and further microscopic analyses were conducted under an optical microscope (Olympus-CX 31, São Paulo, SP, Brazil), coupled to a digital camera (Motic-Motican 3.0 MP). The images were analyzed using Motic software (Images Plus 2.0 ML). The analyses were conducted based on the observation of the filaments and cuts made with the aid of steel blades. Minimum and maximum measurements were taken for morphometric characteristics from a set of 10 measurements for each analyzed structure.
Molecular analysis
Total DNA was extracted after grinding in liquid nitrogen using the NucleoSpin© Plant II-Macherey-Nagel kit (Bethlehem, PA, USA), following the manufacturer’s instructions. For polymerase chain reaction (PCR), the rbcL marker was amplified using three overlapping pairs of primers: FrbcLS-R492a, F492a-R1150a, and F993-RrbcS (Freshwater & Rueness 1994; Cassano 2009). The COI-5P marker was amplified using the primer pair GAZF1-GAZR1 (Saunders 2005). For PCR amplification, two GoTaq kits were used following the manufacturer’s instructions: Flexi DNA Polymerase Kit (Promega, Madison, WI, USA) and G2 Hot Start Polymerase Kit (Promega); the final volume of the reaction was 25 μL. As a way of solving problems in the amplification, bovine serum albumin (0.5 μL) or magnesium chloride (1.5 μL at 25 mM) were added to the PCR solution, always maintaining the final volume at 25 μL. For rbcL, the amplification cycle involved an initial denaturation step at 94 °C for 4 min, followed by 35 cycles of 1 min at 94 °C, 1 min at 45 °C, and 1 min 30 s at 72 °C with a final 10 min extension cycle at 72 °C. For COI-5P, the amplification involved an initial denaturation step at 94 °C for 5 min, followed by 34 cycles of 30 s at 94 °C, 1 min at 45 °C, and 2 min at 72 °C with a final 7 min extension cycle at 72 °C. The reactions were performed using a Techne TC-330 thermocycler (Ramsey, MN, USA). All PCR products were analyzed via electrophoresis on 0.7% agarose gel and stained with Gel Loading Buffer GelRed™ (Biotium) to check product size. PCR products were purified using the PureLinkTM PCR Purification Kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions.
Purified amplicons for both markers were sequenced in both directions using the same PCR primers mentioned above, and the BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA, USA) on an ABI PRISM 3130DNA Genetic Analyzer (Applied Biosystems) using 40 cycles of 10 s at 96 °C, 20 s at 54 °C and 4 min at 60 °C.
The sequences obtained in the forward and reverse directions for each marker were compared using the BlastN algorithm of GenBank (<http://www.ncbi.nlm.nih.gov>), and alignment was performed using BioEdit 7.0.4.1 (Hall 1999). Ambiguous nucleotides were checked in the original electropherograms. For each marker, an alignment was constructed using the sequences generated in the present study and those available in GenBank (Tab. 1).
The most appropriate models of sequence evolution for maximum likelihood (ML) and Bayesian inference (BI), GTR+F+I+G4 for rbcL and HKY+I for COI-5P, were selected based on the Akaike Information Criterion, as implemented on the IQ-Tree webserver (Trifinopoulos et al. 2016). ML analysis was performed using 2,000 bootstrap replicates on the IQ-Tree web server. BI analysis was performed using MrBayes v3.2.2 (Ronquist et al. 2012), with the following parameters: four chains of the Markov chain Monte Carlo (one hot and three cold) for two independent runs, sampling one tree every 1,000 generations for 5,000,000 generations, starting with a random tree. We discarded the first 20,000 generations in both runs as burn-in to build a consensus tree and compute the posterior probabilities. The best tree was visualized using FigTree v1.4.4 (Rambaut 2018). Boldia, Erythrotrichia, and Sahlingia were used as outgroups. For rbcL and COI-5P matrices, genetic distances were calculated using uncorrected ‘p’ distances in PAUP v4.0 beta10 (Swofford 2002).
Results and Discussion
For rbcL, 44 sequences were used in an alignment of 1117 bp, including one new accession obtained in the present study (Tab. 1). The rbcL consensus tree (Fig. 1) containing all global populations of Compsopogon caeruleus sequenced thus far recovered the species as monophyletic with full support. The same was observed for COI-5P, for which 33 sequences (one new accession) were used in an alignment of 664 bp (Fig. 2). The divergence values between our sequences and those from GenBank were 0.13%-1.2% for rbcL and 1.15%-1.48% for COI-5P. Other studies reported similar results, demonstrating low genetic diversity in C. caeruleus samples from around the world (Necchi Junior et al. 2013; Nan et al. 2016).
Consensus tree derived from maximum likelihood (ML) analysis of rbcL sequences. Bootstrap values (BP) and posterior probabilities (PP) are shown at the nodes ML/PP. The sample generated in this study is in bold; ‘-’ indicates lack of bootstrap support or values < 70.
Consensus tree derived from maximum likelihood (ML) analysis of COI-5P sequences. Bootstrap values and Bayesian posterior probabilities (BP/PP) are shown at the nodes. The sample generated in this study is in bold; ‘-’ indicates lack of bootstrap support or values under 70.
Compsopogon caeruleus (Fig. 3a-i) has a densely branched filamentous thallus, with alternate to irregular branching, green to gray in color, and up to 30 cm. Thalli, which is heterotrichous, is composed of pluriseriate main axes and uniseriate lateral branches, corticated in older portions. The pluriseriate portion contains numerous spine-like branchlets. Uniseriate branches are 12-50 µm in diameter. Pluriseriate branches have 1-2 layers of cells, 87-300 µm thick; axial cell is 40-200 µm in diameter. In surface view, cortical cells of the outermost layer are polygonal to irregular, and of the second layer, when it occurs, are globose; cortical cells measure 12.5-50 µm in diameter and 10-50 µm length. Monosporangia present in corticated filaments, spherical to polygonal, and 9-16 µm in diameter.
a-i. Morphological characters of Compsopogon caeruleus from Taperoá, Bahia, Brazil - a. aspect of corticated and uniseriate filaments (scale bar = 200 µm); b. details of uniseriate branches (scale bar = 100 µm); c. aspect of spine-like branchlets (scale bar = 200 µm); d. details of spine-like branchlets (scale bar = 200 µm); e. cross section of an apical corticate filament, showing one layer of cortical cells (scale bar = 50 µm); f. cross section of a corticated filament of the median portion, showing one layer of cortical cells; arrow indicates a monosporagia (scale bar = 20 µm); g. cross section of a corticated filament of the basal portion, showing two layers of cortical cells (scale bar = 50 µm); h. details of a corticated branch of the median portion; arrows indicate monosporangia (scale bar = 100 µm); i. details of a corticated branch of the basal portion; arrows indicate monosporangia (scale bar = 100 µm).
Compared with other studies (for example: Thaxter 1900; Oliveira Filho & Pereira 1973; Necchi Junior et al. 1999, 2013; Bautista & Necchi Junior 2014), few morphological differences exist; however, some studies have highlighted the absence of spine-like branchlets (South & Skelton 2002) and the presence of more layers of cortical cells (Liu & Wang 2004; Ratha et al. 2007; Vis & Necchi Junior 2021). A summary of the comparison of C. caeruleus characteristics from the present study with those from other regions of the world is presented in Table 2.
Comparison of morphological characters of Compsopogon caeruleus from the present and other studies.
The hydrology of the Graciosa region, characterized by the influence of rivers, such as the Rio Graciosa and major tributaries entering the Canal de Taperoá (e.g., Rio do Engenho, Rio das Almas, and Rio Una), generates a highly dynamic estuarine environment (Araujo et al. 2013, Ferraz & Accioly 2015). This pronounced salinity range and the transitional nature of the Graciosa/Taperoá estuarine system provide an ecological context that is consistent with the observed distribution of C. caeruleus. Although primarily considered a freshwater taxon, C. caeruleus has been recorded in estuarine and brackish lagoon environments worldwide. Early studies attested to this brackish water tolerance, reporting the species as “frequent” in tidal creeks within estuarine regions of the United States (Thaxter 1900; Taylor 1960). In India, this species has been collected directly from an estuarine channel and the brackish Chilika Lagoon (Ratha et al. 2007). Kitayana (2011) reported the presence of C. caeruleus along the Pacific coast of Japan, indicating a distribution pattern associated with coastal and estuarine habitats.
Acknowledgements
JMCN: to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brasil, for the Research Productivity Fellowship (308261/2022-4). VC: The São Paulo Research Foundation (FAPESP, 2018/06085-1), for financial support, and CNPq for the Research Productivity Fellowship (304141/2020-8).
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Data availability statement
In accordance with Open Science communication practices, the authors inform that all data supporting the findings of this study are included in the article.
