Introduction

The brown algal genus Coilodesme Strømfelt (Chordariaceae, Ectocarpales) occurs in the cold temperate waters of the Pacific Ocean in the Northern Hemisphere (Deshmukhe 1997). According to Algaebase, 11 accepted species are currently recognized within this genus (Guiry and Guiry 2024). Species of Coilodesm are characterized by the saccate, cylindrical to flattened thalli, larger medullary cells, smaller pigmented cortical cells, a basal disk with rhizoids penetrating the host tissue, and heteromorphic life history that includes a macroscopic sporophyte phase and a microscopic gametophyte phase (Deshmukhe 1997).

In the Northwest Pacific Ocean, several Coilodesme species have been recorded, including Coilodesme cystoseirae (Ruprecht) Setchell et N.L.Gardner, C. japonica Yamada, C. bulligera Strömfelt, and C. fucicola (Yendo) Nagai. These identifications were primarily identified based on thallus size and preferred host species (Okamura 1936, Nagai 1940, Yoshida et al. 1990, Selivanova and Zhigadlova 1997, Kozhenkova 2009). However, taxonomic studies on this genus have been limited to morphological observations, and no phylogenetic analyses have been conducted to date.

This study presents the first report of Coilodesme japonica in Chinese waters, based on molecular phylogenetic analyses using chloroplast-encoded rbcL and mitochondrial cox1 and cox3 gene sequences, combined with morphological diagnoses.

Materials and Methods

Specimens were collected by SCUBA diving from Xicaiyuan (38°21’6”N, 120°54’5”E) and Lingshan (38°21’48”N, 120°54’55”E) of Nanhuangcheng Island, Miaodao Archipelago, China, on June 7, 2024, and August 3, 2024 (Fig. 1). Samples were fixed in a 10% formalin solution for morphological observation, while others were stored at –20°C for DNA extraction and herbarium preparation. All specimens were deposited in the Marine Biological Museum of the Chinese Academy of Sciences (MBM288120-288129). Morphological observations were conducted on manually sectioned samples using a compound microscope. Photographs of the connection points were taken with a digital camera (SONY Alpha 5100, Thailand) mounted on a zoom stereo microscope (Nikon SMZ1500, Japan). Photomicrographs were captured using an upright microscope (Zeiss Imager.Z2, Germany), and the length and width of medullary and cortical cells were measured. Herbarium specimens were prepared and scanned using a scanner (MICROTEK MRS-600A3LED, Shanghai, China).

Fig. 1.

The collection sites (red arrows) on Nanhuangcheng Island, China, in the present study.

Genomic DNA was extracted using a Plant Genomic DNA Kit (Tiangen Biotech, Beijing, China) following the manufacturer's instructions. The rbcL, cox1, and cox3 genes were amplified via PCR using the TaKaRa Ex Taq enzyme in 25-µL reaction volumes (TaKaRa, Japan). The primers used for amplification are listed in Table 1. The newly generated sequences, along with those retrieved from public databases, were aligned using ClustalX and manually adjusted. The terminal regions of the new sequences were trimmed to match those of publicly available sequences. Phylogenetic trees based on rbcL, cox1, cox3 sequences were constructed using the Maximum Likelihood (ML) method as implemented in PhyloSuite software (Zhang et al. 2020), with 1,000 bootstrap replicates to assess the robustness of the tree topology.

Results

Molecular phylogenetic analysis

A total of six new rbcL sequences (1,347bp, PQ658819-658824), four new cox1 (591bp, PV158266-158269), and four new cox3 sequences (488bp, PQ658825-658828) were obtained. In addition, 24 rbcL, 20 cox1 and 15 cox3 related sequences were downloaded from GenBank, with Pylaiella littoralis (Linnaeus) Kjellman included as an outgroup. In the molecular phylogenetic tree based on rbcL sequences (Fig. 2), our specimens formed a distinct clade with those from Hokkaido (Japan) and Kamchatka (Russia), which had previously been identified as Coilodesme japonica. Moreover, the C. japonica clade was found to be sister to Coilodesme californica (Ruprecht) Kjellman, with sequences from Washington (U.S.A.). The phylogenetic tree based on mitochondrial cox1 sequences showed a similar topology (Fig. 3), where our specimens formed a stable clade with C. japonica from the Kamchatka and were also sister to C. californica. However, due to limited availability of cox3 sequences of Coilodesme, only the C. japonica sequences formed a clade with a high bootstrap value in the cox3 tree (Fig. 4).

Fig. 2.

Maximum-likelihood tree based on rbcL gene sequences.

Fig. 3.

Maximum-likelihood tree based on cox1 gene sequences.

Fig. 4.

Maximum-likelihood tree based on cox3 gene sequences.

Morphological analysis

Most specimens of Coilodesme japonica are epiphytic on the vesicles and blades of Sargassum horneri (Turner) C.Agardh at subtidal depths of 3–5 m. The thalli are hollow, saccate, unbranched, long-cylindrical, with a wrinkled surface, light brown in color, and measure 4.7–40 cm in length and 0.5–3.5 cm in width (Figs. 5-6). The thallus grows on the surface of Sargassum horneri, attached by a small discoid holdfast (Fig. 7). Irregular cortical cells, observed from the surface view, measure 9.61 ± 1.42 µm (Fig. 8). The basal part of the thallus of C. japonica is embedded in the apex of a blade of S. horneri, with rhizoids penetrating the host tissue (Fig. 9). The cortex consists of 2–3 layers of smaller pigmented cells, measuring approximately 12.4 ± 2.43 µm in length and 13.28 ± 2.73 µm in width. The medulla comprises 1–2 layers of colorless cells, approximately 105 ± 52.59 µm in length and 32.8 ± 12.75 µm in width in transverse section (Fig. 10). Unilocular sporangia are embedded in the cortical layer, measuring 17–23 × 12–16 µm (Figs. 11-12).

Fig. 5.

Plants growing underwater; Fig. 6. The herbarium (MBM288120) collected from Nanhuangcheng Island, showing hollow saccate, long-cylinder-like thalli attaching on the host Sargassum horneri.

Fig. 7. The basal part of a thallus of C. japonica (arrow heads) connected to a blade of S. horneri (arrow); Fig. 8. Surface view of cortical cells; Fig. 9. Cells of C. japonica (arrow heads) embedded in cells of S. horneri; Fig. 10. Transverse section of the middle portion of a thallus, showing smaller cortical and larger medullary cells; Fig. 11. Unilocular sporangium embedded in cortical layer (arrow); Fig. 12. Longitudinal section of a mature thallus, showing unilocular sporangium in cortical layer (arrow).

Fig. 7.

The basal part of a thallus of C. japonica (arrow heads) connected to a blade of S. horneri (arrow); Fig. 8. Surface view of cortical cells; Fig. 9. Cells of C. japonica (arrow heads) embedded in cells of S. horneri; Fig. 10. Transverse section of the middle portion of a thallus, showing smaller cortical and larger medullary cells; Fig. 11. Unilocular sporangium embedded in cortical layer (arrow); Fig. 12. Longitudinal section of a mature thallus, showing unilocular sporangium in cortical layer (arrow).

Discussion

The molecular phylogenetic trees, constructed using both chloroplast and mitochondrial gene sequences, confirm the monophyletic nature of the genus Coilodesme. Our results suggest that our specimens and those from the type locality of C. californica (Muroran, Hokkaido, Japan) are conspecific, as their rbcL and cox3 gene sequences were found to be 100% identical. Furthermore, C. californica is closely related to the C. japonica clade, with both species clustering together with high bootstrap support in the phylogenetic trees based on rbcL and cox1 gene sequences. Unfortunately, due to the limited availability of molecular data, the phylogenetic relationships of C. japonica with most species of Coilodesme, as well as with other genera in the family Chordariaceae, remain unclear.

Morphologically, Coilodesme japonica differs from the most phylogenetically related C. californica, by its smaller size, thinner cortex, and fewer medullar layers. This species is also distinguished from other Coilodesme species based on differences in thallus morphology, the number of cortical and medullary layers, and the size of sporangia (Table 2). As previously reported, C. japonica is predominantly found growing on Cystosiera hakodatensis (Yendo) Fensholt and Sargassum confusum C. Agardh in Hokkaido, Japan (Yamada 1938, Deshmukhe and Tatewaki 2001). The species has also been identified as a parasite of the Kelp Alaria Greville in Kamchatka, Russia, based on high-throughput sequencing (Bringloe et al. 2021). Interestingly, C. japonica was not detected on any other local macroalgae from the collection sites, except for S. horneri.

The seaweed diversity is high in the waters surrounding small offshore islands in the northern China seas, largely due to better water transparency and nutrient availability in the region. More than 300 species have been recorded (Tseng 2009, Luan 2013), however, the brown alga Coilodesme japonica was never previously reported. Additionally, no taxonomic documents or deposited herbarium specimens of this species have been found to date. We first identified small thalli of C. japonica attached to Sargassum horner in the summer of 2020 at Lingshan, Nanhuangcheng (Fig. 1), and it has not been observed in other waters around the island.This suggests that C. japonica has not been present in these waters for a long time. Although mature thalli on S. horneri can drift over long distances, they do not appear to easily establish in new habitats where summer water temperature exceed 26°C. The population may settle down and expand after years or decades, similar to other cold-water invasive algae, such as Costaria costata (C.Agardh) De A.Saunders, Saccharina japonica (Areschoug) C.E.Lane, C.Mayes, Druehl et G.W.Saunders (Zheng et al. 2024).

The advent of globalization has facilitated the transport of algal species beyond their native range, primarily through attachment to ship hulls and ballast water. Additionally, the intentional or unintentional introduction of aquatic animals and plants has contributed to species invasions (Williams and Smith 2007). As a potentially invasive species, the population expansion and associated ecological impact of C. japonica warrant further investigation.