Colaconema zhanjiangensis sp. nov. (Colaconemataceae, Rhodophyta): a new epi-endophytic filamentous red algal species from China

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Abstract

INTRODUCTION

The marine red algal genus Colaconema Batters (Colaconemataceae, Rhodophyta) currently comprises 52 species that are widely distributed in tropical and temperate regions (Guiry and Guiry 2025). Colaconema, established by Batters, was originally classified within the order Acrochaetiales Feldmann but was transferred to the newly established order Colaconematales J. T. Harper & G. W. Saunders in 2002 (Harper and Saunders 2002, Hwang and Kim 2011). This reclassification clarified the systematic position of several previously misidentified species. Although members of the Colaconematales generally have more elongated cells than those of the Acrochaetiales, their high morphological similarity continues to complicate species-level identification (Harper and Saunders 2002, Hwang and Kim 2011). Colaconema species are characterized by uniseriate filaments composed of cylindrical cells containing one or more parietal plastids-lobed, laminar, spiral, or irregular, but not stellate, with or without pyrenoids. Monosporangia are usually produced on terminal or intercalary cup-shaped cells, and reproduction is primarily asexual by monospores, with a triphasic life history (Harper and Saunders 2002, Santos and Moura 2010, Hwang and Kim 2011, Lee and Yeh 2021). However, morphological features are often plastic and environmentally influenced, causing difficulty in species delimitation (Garbary 1979, Soares et al. 2020). Consequently, molecular-assisted taxonomy has become essential for distinguishing morphologically similar taxa, particularly using rbcL and 5′ region of cytochrome c oxidase subunit I (COI-5P) gene markers (Araújo et al. 2014, Montoya et al. 2020).

Although Colaconema species have been reported worldwide, studies in China remain limited, with species identification relying primarily on morphological characteristics and relatively few species confirmed using molecular markers. A total of 12 species of Colaconema have been recorded based on taxonomic studies from Mainland China and Taiwan, China. Among these, only C. formosanum M. C. Lee & H. Y. Yeh has been confirmed by molecular analysis (Tseng 2005, Titlyanov et al. 2017, Lee and Yeh 2021, Guiry and Guiry 2025). This gap underscores the need for further molecular studies to accurately resolve species identities and to support the taxonomic framework of the genus. Notably, several of these species are recorded in Audouinella Bory and Acrochaetium Nägeli in Chinese taxonomic literature, reflecting the taxonomic confusion within the group (Tseng 2005, Titlyanov et al. 2017). Such taxonomic uncertainty, coupled with the lack of molecular support for species identifications, limits our understanding of Colaconema diversity and its evolutionary relationships.

Globally, Colaconema species occur as epiphytes, endophytes, or epi-endophytes on macroalgae such as Sargassum C. Agardh, Gelidium J. V. Lamouroux, and Polysiphonia Greville (Hwang and Kim 2011). Solieria J. Agardh (Solieriaceae, Rhodophyta) is an economically important macroalgal genus and one of the major carrageenan-producing taxa, widely used in the food, pharmaceutical, and aquaculture industries (Stephanie et al. 2010, Chopin and Tacon 2021, Burlot et al. 2023). During the cultivation of Solieria sp., an unidentified epi-endophytic red alga was observed proliferating extensively on its thalli, causing necrosis and loss of elasticity. However, no epi-endophytic Colaconema species associated with Solieria have been documented in China to date. In this study, we characterize an epi-endophytic Colaconema species isolated from Solieria sp. in Zhanjiang, Guangdong Province, China, based on detailed morphological observations and molecular analyses of the chloroplast rbcL and mitochondrial COI-5P genes. This finding contributes to the taxonomic and molecular understanding of Colaconema diversity.

MATERIALS AND METHODS

DNA extraction, amplification, and phylogenetic analyses

DNA was extracted from 50–100 mg of fresh red filamentous algae using the Plant Genome Rapid Extraction Kit (Tiangen Biotech, Beijing, China) following the manufacturer’s instructions. An additional chloroform-based phase separation step was performed to remove polysaccharides, proteins, and other contaminants. The quality and concentration of the resulting DNA were evaluated spectrophotometrically.

The large subunit of rbcL was amplified with the primer pairs F57-R1381, F7-R753 and F645-RrbcLstart (Table 1) (Freshwater and Rueness 1994, Lin et al. 2001, Gavio and Fredericq 2002). The mitochondrial COI-5P gene was amplified by polymerase chain reaction (PCR) using the primers GazF1 and GazR1 (Table 1) (Saunders 2005, Montoya et al. 2020). The PCR protocol consisted of an initial denaturation at 94°C for 3 min, followed by 35 cycles conducted under primer-specific conditions: F57-R1381, 30 s at 94°C, 40 s at 49.5°C, and 90 s at 72°C per cycle; F7-R753, 30 s at 94°C, 40 s at 50°C, and 60 s at 72°C per cycle; F645-RrbcLstart, 60 s at 94°C, 60 s at 45°C, and 60 s at 72°C per cycle; GazF1-GazR1, 30 s at 94°C, 40 s at 50°C, and 50 s at 72°C per cycle. All reactions were completed with a final extension at 72°C for 5 min, followed by a soaking step at 4°C. PCR conditions, including primers, annealing temperatures, and extension times, are listed in Table 1. Products were verified by 1% agarose gel electrophoresis and sequenced commercially (BGI, Guangzhou, China). Commercial sequencing is based on the principle of the Sanger method with double deoxy chain end termination. DNA sequences of rbcL and COI-5P genes were obtained from the unknown epi-endophytic alga. All new sequence data generated from this study were deposited in GenBank, with the following accession numbers: PQ461390–PQ461393 (rbcL) and PQ461386–PQ461389 (COI-5P).

Table 1

Polymerase chain reaction (PCR) primer sequences

For rbcL analysis, we compiled sequence data available from GenBank for 7 Colaconema species and 8 orders belonging to Nemaliophycidae: Palmariales (n = 7), Acrochaetiales (n = 5), Batrachospermales (n = 7), Entwisleiales (n = 2), Nemaliales (n = 6), Ottiales (n = 2), Rhodachlyales (n = 2), and Thoreales (n = 6). For COI-5P analysis, we compiled sequence data available from GenBank for 5 Colaconema species and 4 orders belonging to Palmariales (n = 6), Acrochaetiales (n = 10), Nemaliales (n = 3), and Batrachospermales (n = 1). Phylogenies were inferred and presented as unrooted because no explicit outgroup taxon was designated. The rbcL and COI-5P sequences were subjected to maximum likelihood (ML) and Bayesian inference (BI) phylogenetic analyses. Sequences were aligned using multiple sequence alignment, manually checked, and trimmed using MEGA X (Kumar et al. 2018). The best-fit model of evolution was selected using IQ-TREE2 (Minh et al. 2020) based on the Bayesian information criterion and Akaike information criterion. The best-fit models for rbcL and COI-5P sequences were GTR + F + I + G4 and TIM2 + F + I + G4, respectively. ML phylogenetic analysis was performed in IQ-TREE, with statistical support assessed using 1,000 bootstrap replicates. The BI was conducted with MrBayes v3.2.7 (Ronquist et al. 2012) applying the GTR + F + I + G4 model for both markers, as TIM2 is not implemented in MrBayes. Two independent runs of four Markov Chain Monte Carlo chains were performed, with trees sampled every 100 generations for a total of 1,000,000 generations. The first 25% of the sampled trees were discarded as burn-in, and the remaining trees were used to calculate the Bayesian posterior probability for each clade. ML and BI trees were visualized and edited in FigTree v1.4.4 software (Rambaut 2018). Genetic distances between sequences were calculated using the Kimura 2-parameter model in MEGA X.

RESULTS

Vegetative structure

The thallus is epi-endophytic on Solieria sp. (Fig. 1 A), bright red color, caespitose, composed of basal discs and erect filaments (Fig. 1 B & C). Erect filaments arise from small basal discs (Fig. 1 C & D). Upon attachment, it can grow to about 5 mm, whereas under isolated culture conditions, it can reach up to 2.5 cm. Endophytic cells invade the cortical and subcortical cells of the host (Fig. 1 D & E). Cells of the erect filaments are cylindrical, not constricted at the septum, while endophytic cells are irregular in shape (Fig. 1 E). Cells with a single pyrenoid, and single parietal or lobed plastids (Fig. 1 F). Branching of erect axes is irregular, with secondary or alternate branches arising at various intervals (Fig. 1 B). Lateral branches typically emerge from the sides of cells along the main axis, forming distinct branches; multiple lateral branches may occasionally arise from a single cell. Lateral branches are generally shorter than the main axes and do not form pseudo-hairs (Fig. 1 B). Cells at the lower base measure 6–10 μm in width and 16–28 μm in length. Cells of erect filaments are 6–8 μm wide and 23–26 μm long, while lateral branch cells are smaller, measuring 5–8 μm in width and 12–22 μm in length. Cells toward the apices are smaller and appear to bear transparent hairs, which are artifacts of the apical cell shape (Fig. 1 J). The length-to-diameter (L:D) ratios of cells at the lower base, mid-branch, and lateral branches are 3.4:1, 3.6:1, and 2.7:1, respectively.

Fig. 1

Morphology of Colaconema zhanjiangensis sp. nov. (A) Thallus epi-endophytic on Solieria sp. (B) Branching of erect axes irregular, primarily secund, with the lateral branches typically being shorter (arrow). (C) The erect filaments (arrowhead) epiphytically grow on the surface, forming a basal disc (arrow). (D) Longitudinal section of the basal disc, a single layer of cells (arrow), erect filaments (arrowhead). (E) Endophytic filaments invade the cortical and subcortical cells (arrow). (F) Detail of erect filaments showing cells with one pyrenoid (arrow). (G) Empty shells (arrow). (H) Sporangia with pedicels and sporangium branches, the arrow indicates a monosporangium. (I) Mature monosporangia. (J) Tapering slightly toward apices (arrow). (K) A tube-like cell emerged from the spore, gradually expanded and branched. (L) Tube-like cell branches and gives rise to erect filaments (arrow). Scale bars represent: A, 5 mm; B, 100 μm; C–E & J, 30 μm; F–H, K & L, 20 μm; I, 10 μm.

The phylogenetic tree analysis of Colaconema species

The rbcL and COI-5P sequences were subjected to ML and BI phylogenetic analyses. A total of 49 rbcL sequences were analyzed, including four newly generated sequences (PQ461390–PQ461393; 1,198 bp), and 34 COI-5P sequences were included, among which four were newly generated in this study (PQ461386–PQ461389; 583 bp). Phylogenetic trees were reconstructed using ML and BI, showing that the unknown specimens’ rbcL and COI-5P sequences nested within Colaconematales, forming a monophyletic clade with distinct affiliations. Because the ML and BI topologies were nearly identical, the ML consensus tree is presented, with BI posterior probabilities mapped onto the corresponding nodes.

In the rbcL phylogeny (Fig. 2), C. zhanjiangensis formed a sister relationship with C. formosanum (MW182307 and MW182308), with strong support (ML = 86%, BI = 1.00). The genetic distance between the two species was 5.06–5.15% (Supplementary Table S1). In the COI-5P phylogeny (Fig. 3), C. zhanjiangensis was also sister to two specimens of C. formosanum (MW182304 and MW182305), again with strong support (ML = 94%, BI = 1.00). The genetic distance between them was 7.23% (Supplementary Table S2). Collectively, the molecular evidence strongly supports the taxonomic placement of this filamentous red algal species from Zhanjiang. We therefore designate it as a new species, Colaconema zhanjiangensis sp. nov.

Fig. 2

Maximum likelihood (ML) topology based on rbcL DNA sequences. ML bootstrap support values are shown to the left of each node, andBayesian posterior probabilities to the right. Sequences generated in this study are shown in bold; otherwise, they are from GenBank.

Fig. 3

Maximum likelihood (ML) topology based on 5′ region of cytochrome c oxidase subunit I (COI-5P) DNA sequences. ML bootstrap supportvalues are shown to the left of each node, and Bayesian posterior probabilities to the right. Sequences generated in this study are shown in bold; otherwise, they are from GenBank.

DISCUSSION

In the present study, C. zhanjiangensis exhibited typical characteristics of the genus, including cylindrical cells, irregularly branched filaments, monosporangia, a single parietal chloroplast, and a single pyrenoid. The species displayed a Type II germination pattern, in which the protoplast remains within the spore during germ tube development (Harper and Saunders 2002). This pattern is consistent with C. daviesii (Dillwyn) Stegenga and C. formosanum and is considered a supplementary diagnostic character for the genus (Montoya et al. 2020, Lee and Yeh 2021). However, data regarding such a character remain scarce across known species, highlighting the need for further research to understand its biological significance.

A total of 12 Colaconema species have been documented from mainland China and Taiwan, China, and a detailed comparison of their morphology, together with that of related Asian taxa, is provided in Table 2 (Tseng 2005, Hwang and Kim 2011, Titlyanov et al. 2017, Lee and Yeh 2021, Guiry and Guiry 2025). C. zhanjiangensis can be readily distinguished from its congeners by a unique combination of morphological characters, including relatively long thalli (up to 5 mm), the presence of a basal disc, medium-sized cylindrical vegetative cells (6–8 μm in diameter and 23–26 μm in length) with a single pyrenoid, and medium-sized ovoid to ellipsoid monosporangia that occur singly, in pairs, or in clusters, either sessile or borne on 1–4-celled pedicels. This suite of characters clearly separates the species from all previously described taxa. C. bonnemaisoniae Batters, C. emergens (Rosenvinge) R. Nielsen, C. nakamurae Woelkerling, and C. robustum (Børgesen) Huisman & Woelkerling differ from C. zhanjiangensis in basal structures, such as having entirely prostrate filaments or a single basal cell, as well as in monosporangial arrangement. C. codicola (Børgesen) Stegenga, J. J. Bolton & R. J. Anderson is characterized by 2–5 pyrenoids per cell and relatively large vegetative cells, in contrast to C. zhanjiangensis, which has a single pyrenoid and medium-sized cells. Similarly, the vegetative cells of C. daviesii, C. dictyotae (Collins) I. K. Hwang & H. S. Kim, and C. thuretii (Bornet) P. W. Gabrielson are considerably larger than those of C. zhanjiangensis. C. codii (Hamel) I. K. Hwang & H. S. Kim has notably larger monosporangia than C. zhanjiangensis, but its vegetative cells are distinctly shorter. Regarding thallus height, C. zhanjiangensis is of medium stature, whereas species such as C. attenuatum (Rosenvinge) R. Nielsen, C. comptum (Børgesen) I. K. Hwang & H. S. Kim, and C. hyalosiphoniae (Nakamura) I. K. Hwang & H. S. Kim have relatively short thalli (≤1.5 mm), and C. formosanum is much taller, reaching 2.0–2.8 cm. The ovoid to ellipsoid monosporangia of C. zhanjiangensis also differ from the subspherical monosporangia of C. formosanum. Overall, the combination of thallus length, basal disc, thallus height, cell and monosporangia size, single pyrenoid, and monosporangial arrangement provides a clear and reliable basis for distinguishing C. zhanjiangensis from all other known Colaconema species.

Table 2

Comparison of diagnostic characteristics between Colaconema zhanjiangensis sp. nov. and other similar Colaconema species distributed across Asia and Southeast Asia

Although the characteristics mentioned are sufficient to distinguish C. zhanjiangensis as a new species, morphological characters, while distinguishing, are often subjective and prone to variable interpretation. Additionally, environmental factors influence algal morphology, further complicating their use in taxonomic evaluation (Freshwater et al. 2022). In contrast, molecular markers have been widely adopted in species identification due to their greater stability and reliability. Among these, the mitochondrial-encoded cox1 gene and the plastid-encoded rbcL gene have been proposed as potential barcodes for rhodophytes (Tan et al. 2012). Consequently, molecular phylogenetics provides a more reliable approach for taxonomy within Nemaliophycidae, with molecular-assisted identification using plastid-encoded rbcL and COI-5P playing a significant role in distinguishing species within Colaconema (Lee and Yeh 2021).

In the rbcL and COI-5P genes analysis, the genetic sequence divergence between C. zhanjiangensis and C. formosanum is 5.06–5.15% and 7.23%, respectively. The genetic distance between C. zhanjiangensis and other species within the Colaconema genus shows that the rbcL gene has a genetic distance ranging from 7.73 to 13.65%, and the COI-5P gene has a genetic distance ranging from 9.80 to 13.09%. There are a few molecular classification reports on Colaconema, but other related studies can provide some reference value. For example, the pairwise genetic distances among related species in the phylogenetic analyses for rbcL were 4.65% between A. secundatum and A. plumosum, and 4.39% between Rhodophysema elegans and R. georgei (Supplementary Table S1). For COI-5P, the distances were 5.54% between A. moniliforme and A. catenulatum, and 6.56% between A. moniliforme and A. densum (Supplementary Table S2). Additionally, it has been reported that the rbcL genetic distance among different Gracilaria species ranges from 3.13 to 9.5% (Wang et al. 2023), while the COI-5P genetic distance among Gelidium species ranges from 2.7 to 5.4% (Boo and Kim 2020). Although these thresholds cannot be directly applied to Colaconema, the relatively conserved evolutionary rates of rbcL and COI-5P across Rhodophyta make them meaningful reference points for assessing species boundaries. Overall, the genetic divergences observed between C. zhanjiangensis and C. formosanum provide sufficient evidence to support the recognition of C. zhanjiangensis as a distinct new species.

Species within the Colaconema genus have potential commercial value. C. formosanum is rich in phycoerythrin, whose purified products exhibit good light stability in the development of UV protection cosmetics, providing an important reference for the development of the newly discovered species in this study (Lee et al. 2021b, Yeh et al. 2022, 2023). Additionally, R-phycoerythrin from Colaconema sp. can modulate the immune response of white leg shrimp, both in vitro and in vivo, indicating its potential as an immunomodulator (Lee et al. 2021a). The protein hydrolysates extracted from C. formosanum exhibited significant antioxidant activity, and the angiotensin-converting enzyme inhibitory peptides from the same species may serve as potential candidates for the development of functional foods for hypertension management (Windarto et al. 2024a, 2024b). Despite limited research on Colaconema species, these findings indicate their potential applications in cosmetics, functional foods, and immunology.

In Solieria sp., infestation led to the formation of dense filamentous masses that enveloped the host thalli. As the infestation progressed, filament proliferation caused tissue softening, fragmentation, and structural collapse. Histological observations confirmed that the filaments had penetrated beyond the epidermis into deeper tissues. These symptoms are consistent with previously reported Colaconema infestations (Vairappan et al. 2008, Araújo et al. 2014, Montoya et al. 2024). Studies have shown that in K. alvarezii infected with Colaconema species, distinct morphological and cellular alterations occur, including disruption of chloroplast structure, reduction of grains, and noticeable thickening of the cell walls (Araújo et al. 2014). The symptoms and cellular damage collectively suggest that Colaconema infestations can lead to host mortality and compromise cultivation. As such, these Colaconema species pose a potential risk to the macroalgal aquaculture industry (Ward et al. 2020, Behera et al. 2022, Montoya et al. 2024).

C. zhanjiangensis, the first Colaconema species reported to infect Solieria in China, is distinguished by distinct morphological characteristics and supported by molecular phylogenetic evidence. Its observed impacts on host health suggest potential risks to macroalgal aquaculture, highlighting the need for ongoing monitoring and additional studies on host specificity, infestation mechanisms, and ecological consequences.

This study reports the first Colaconema species infecting Solieria in China. Both morphological and molecular evidence support its recognition as a new species, Colaconema zhanjiangensis sp. nov. We recommend that future studies employ metabolomics approaches to investigate changes in secondary metabolites during host-parasite interactions, which may help elucidate parasitic mechanisms and provide insights relevant to potential biocontrol strategies. Furthermore, we encourage researchers to deposit molecular data of other Colaconema species in public databases, which would facilitate more accurate and comprehensive classification and promote global collaboration in the systematics of this genus.

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