Journal of Oceanology and Limnology   2022, Vol. 40 issue(6): 2343-2353     PDF       
http://dx.doi.org/10.1007/s00343-022-1392-y
Institute of Oceanology, Chinese Academy of Sciences
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Article Information

WEI Xiu, LIU Wenzheng, LIN Xuyin, LIU Qianchun, JIANG Peng
First record of Ulva californica in the mainland of China: a single alien parthenogenetic population in discontinuous distribution
Journal of Oceanology and Limnology, 40(6): 2343-2353
http://dx.doi.org/10.1007/s00343-022-1392-y

Article History

Received Nov. 17, 2021
accepted in principle Dec. 22, 2021
accepted for publication Feb. 25, 2022
First record of Ulva californica in the mainland of China: a single alien parthenogenetic population in discontinuous distribution
Xiu WEI1,2,3, Wenzheng LIU2,3,4, Xuyin LIN5, Qianchun LIU2,3,4, Peng JIANG2,3     
1 College of Life Science, Qingdao University, Qingdao 266071, China;
2 CAS and Shandong Province Key Laboratory of Experimental Marine Biology, Center for Ocean Mega-Science, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China;
3 Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology(Qingdao), Qingdao 266237, China;
4 University of Chinese Academy of Sciences, Beijing 100049, China;
5 Xiamen Ocean Vocational College, Xiamen 361012, China
Abstract: Molecular investigations have raised concerns about the ecological risks of green tides caused by alien Ulva species in new habitats. The green tide-forming species U. californica Wille was generally considered to be native to North America, but new records have been widely reported in Europe, Asia, and Oceania in recent decades, indicating a strong dispersal capacity of the species. In this study, the first record of U. californica on the coastline of mainland China was reported, following a combined identification with multi-molecular markers and morphological characterization. It was shown that this species has a discontinuous distribution pattern along the coast of mainland China, with northern populations in the Yellow Sea and southern populations in the East China Sea and South China Sea. According to results of examination for life cycles and identification with mating type (MT) genetic markers, it was indicated that all U. californica samples were male gametophytes, and reproduced themselves through parthenogenesis solely. Combined with the fact that southern and northern populations are highly genetically identical, here we believed that U. californica was a recent alien species to mainland China with a rapid local spread. This finding provided evidences that the ability to reproduce in a variety of ways may play an important role in the spread of Ulva species, as well as essential basic data for marine risk management of green tides in China. In addition, according to the phylogeographic analysis, the possible geographical origin and global dispersal routes of U. californica were also proposed.
Keywords: discontinuous distribution    mainland of China    new record    non-indigenous species    parthenogenesis    Ulva californica    
1 INTRODUCTION

The species of green algal genus Ulva Linnaeus are widely distributed all over the world. They are found in a wide range of habitats from brackish coast to freshwater areas, and thrive in both life patterns, i.e., either being attached to different substrates or floating freely (Norris, 2010). Many Ulva species are typical opportunistic seaweeds that they can grow rapidly once the temperature is suitable and the nutrients are abundant, sometimes causing massive macroalgal blooms and serious economic losses (Fletcher, 1996; Smetacek and Zingone, 2013). Therefore, the ability to accurately identify Ulva species becomes very important. However, morphological identifications of Ulva species are notoriously difficult due to the lack and instability of distinguishable features (Blomster et al., 2002; Wichard, 2015), resulting in serious misapplication of species names to a large extent (Hughey et al., 2021). Instead, the development of molecular techniques has made important contributions to the clarification of species identification in Ulva (Hayden et al., 2003).

Recently, molecular-based investigations of Ulva biodiversity have been widely carried out globally, resulting in the discovery of quite a few new species, cryptic species, or alien species. For example, several new species including U. ohnoi were identified in Japan (Hiraoka et al., 2004); U. chaugulii and U. tepida were recorded for the first time in the Mediterranean (Krupnik et al., 2018); and U. australis (syn. U. pertusa) was detected as an introduced species to the Pacific coast of Mexico (Aguilar-Rosas et al., 2008) and North Atlantic (Hofmann et al., 2010). It should be noted that, some of new record or introduced species were found to cause green tides locally, such as U. tepida as a new record in India (Bast et al., 2014), U. ohnoi as a non-native species in the Gulf of Mexico and Atlantic Florida (Melton III et al., 2016), and U. meridionalis, U. tepida, U. chaugulii as alien species in the South China Sea (Xie et al., 2020). Therefore, these facts suggested that it is necessary to strengthen the monitoring of new records or alien species of Ulva, which is the key for risks evaluation of green tides.

Ulva californica Wille was firstly described in 1899 in type location at La Jolla, California (Collins et al., 1899). According to the comparisons on morphological and developmental characters, Tanner (1986) placed both U. angusta Setchell & N.L.Gardner (= Enteromorpha angusta (Setchell & N.L.Gardner) Doty) and U. scagelii Chihara as taxonomic synonyms of U. californica. Despite their thalli differed in shape and size from those of U. californica, all three taxa had similar developmental patterns including the formation of germination tubes. This taxonomic revision was later confirmed by Hayden and Waaland (2004) using molecular methods, and the occurrence of U. californica in Europe was also detected after a reassessment of previously published molecular data (Tan et al., 1999). In addition, based on molecular identification and hybridization experiments, a clear species boundary among U. californica, U. flexuosa, and a closely related species U. mediterranea, which was later renamed as U. aragoënsis (Krupnik et al., 2018), was set up (Hiraoka et al., 2017). Although opinions on the original habitat of U. californica varied, with some claiming it was restricted to North America (Scagel et al., 1989; Wolf et al., 2012), others suggesting a larger distribution area (Loughnane et al., 2008), the introductions of this species to many new habitats have been widely noticed (Kawai et al., 2007; Heesch et al., 2009; Wolf et al., 2012; Kirkendale et al., 2013). In China, except for few early morphological records of U. angusta in Taiwan (Okamura, 1935; Chiang, 1973), the occurrence of this species has never been described in the mainland of China (Ding et al., 2015).

In this study, samples of U. californica were collected from the coastal mainland China for the first time. Molecular and morphological approaches were combined for species identification. The observation on life cycle with the detection of genetic markers for mating type (MT) was carried out to reveal the reproduction mode. Additionally, phylogeographic analysis was performed to predict the possible genetic origin of U. californica samples. Since U. californica was reported as a green-tide-forming species that has ever bloomed in the coastal zone of Jeju Island (Bae, 2010), this study may provide essential basic data for marine risk management in China.

2 MATERIAL AND METHOD 2.1 Seaweeds and culture conditions

A total of 29 blade-like samples of Ulva, later identified by molecular identification as U. californica Wille, were collected from eight coastal sites in the Yellow Sea, East China Sea, and South China Sea from 2020 to 2021 (Fig. 1; Supplementary Table S1). All samples were transported back to laboratory in a cold container for further research, and then washed in sterilized seawater to remove any contaminants and epiphytes. Each thallus was placed individually in a dish with Von Stosch's Enriched medium (VSE medium) which was renewed every 3–4 days. All seaweeds samples were cultured at 16 ℃ with 12-h:12-h light (L):dark (D) photoperiod and illumination intensity of 80–100 μmol photons/ (m2·s).

Fig.1 Sampling sites of Ulva californica along the coastline of China Black dot indicates the sampling sites.
2.2 Molecular identification

From each individual, approximately 1 cm2 of thallus was cut off to extract the total genomic DNA with a Plant Genomic DNA Extraction Kit (Tiangen Biotech Co., Ltd., Beijing, China), according to the manufacture's protocol. Three pairs of primers for the internal transcribed spacer (ITS) (Leskinen and Pamilo, 1997), the large subunit of ribulose-1, 5-bisphosphate carboxylase/oxgenase (rbcL) (Manhart, 1994), and the chloroplast elongation factor (tufA) (Saunders and Kucera, 2010), were combined to use for species identification. All the profiles of PCR ampification followed the previous descriptions (Xie et al., 2020). The PCR products were visualized on 1.5% agarose gel stained with Super GelRed (US Everbright Inc., Suzhou, China), PCR products were purified and then sequenced by Ruibio BioTech Co. Ltd, Qingdao, China. The phylogenetic analysis was carried out with the DNA sequences from both samples and those downloaded from the GenBank database, plus the homologous sequences from Ulvaria or Blidingia as outgroups. The maximum likelihood (ML) phylogenetic trees were constructed using MEGA 6.0 (Tamura et al., 2013), with 1 000 bootstrap replicates.

2.3 Morphological characterization

After molecular identification and phylogenetic analysis, healthy and intact thalli of U. californica samples were selected to prepare the seaweed specimen, which was deposited in the herbarium of the Institute of Oceanology, Chinese Academy of Sciences (IOCAS). Both the gross morphology and anatomical features of U. californica were characterized, including the shape and size of thalli; the shape and arrangement of the surface cells; the pyrenoid number per cell; and the transverse section view of thalli. The vegetative thalli were stained with Lugol's iodine for counting the pyrenoids inside the cells. Microscopic observations were carried out using a BH2 light microscope (Olympus Corp., Tokyo, Japan), and photographs were taken using CCD camera (Scope Tek MDC200, Mingshi, Ningbo, China).

2.4 Reproduction, life cycle, and morphogenesis

To determine the life history type of selected population samples, the formation of germ cells was induced within 4–6 days by excising small fragments (1–5-mm long) from healthy thalli (Hiraoka and Enomoto, 1998). The number of germ cells in each germ cell sac was investigated. Freshly released germ cells were pipetted into new Petri dishes and cultured under same conditions in VSE medium. The phototaxis of germ cells was recorded, and the flagella were stained with Lugol's iodine and counted under the light microscope. Considering that only using phototaxis and number of flagellum to determine the nature of germ cells were not completely reliable (Hiraoka et al., 2003; Matsumoto and Shimada, 2015), a pair of universal degenerate primers, mating type minus (mt-) PRA1m and mating type plus (mt+) PRA1f which have been developed for the identification of MT loci in Ulva, were used. This pair of primers can amplify products from both male and female MT regions, which were divergent in sequence between two sexes. The PCR reactions were performed with both the mother thalli, which were selected for induction of reproduction, and the progeny generated from the released germ cells, following the protocol described in this literature (Yamazaki et al., 2017). And the procedures for sequencing of PCR products and phylogentic analysis were as same as mentioned above. Furthermore, the entire period of morphogenesis was recorded from the germination of germ cells to the formation of adult thalli.

2.5 Biogeographic and phylogeographic analysis

To analyze the global biogeographic distribution pattern of U. californica, all highly similar sequences (identity ≥98% for ITS or tufA, and identity ≥99% for rbcL) were downloaded from the GenBank database. In addition, all homologous sequences that have been annotated as those of U. californica for each marker were also included. After phylogenetic analysis and reassessment of species identity, the sampling location information for each selected sequence was extracted for further bio- and phylo-geographic analysis.

3 RESULT 3.1 Molecular identification

A total of 55 nucleotide sequences of rbcL, including 29 samples from this investigation and 26 reference sequences downloaded from GenBank, were used for species identification and phylogenetic analysis. As shown in Fig. 2 using TN93+G model, all the samples were gathered into an U. californica cluster, which contains reference sequences of those collected from type location in California, and obviously separated from other species including the genetically closest species U. aragoënsis. Two other ML phylogenetic trees based on tufA or ITS respectively showed similar topological structures to that of the rbcL-ML tree (Supplementary Figs.S1 & S2). Therefore, it can be confirmed that all the 29 samples belong to U. californica. It was worth noting that all samples shared exactly identical sequences of rbcL or tufA markers.

Fig.2 Maximum likelihood (ML) phylogenetic tree based on DNA sequences of rbcL Numbers at the nodes indicate bootstrap values. GenBank accession numbers for all reference sequences and information of sampling locations for U. californica reference sequences are provided. Sequences in bold were from samples in this study. Numbers in parentheses following the sample ID represent the amount of identical sequences.
3.2 Morphological characterization

Samples of U. californica exhibited a variety of shapes, from lanceolate to amorphous. The thalli were distromatic sheets and usually bright green, with a ruffled margin and rough surface. The samples from the East China Sea and South China Sea were 2–25 cm in height and 1–4 cm in width (Fig. 3a–c), while those from the Yellow Sea were 5–7 cm in height and about 1 cm in width (Fig. 3d). In surface view, the cells were 4–6 sides, angular or with rounded corners. Each cell contained one chloroplast, which filling the whole cell (Fig. 3e). The cells are usually arranged closely and formed a regular row. The average sizes of the middle region cells were 17.2±3.5 μm in length and 11.2±2.3 μm in width (Fig. 3e). In transverse section, the blades consisted of two layers of cells which were rectangular with rounded corners, and the chloroplasts were positioned towards the outside of the blade, and the thickness were 61.3±2.5 μm (Fig. 3f) (n=20). After staining with Lugol's iodine, each cell was shown to possess 1–3 pyrenoids, and most cells only had one pyrenoid (one, 77.5%; two, 21.2%; three, 1.3%; n=316) (Fig. 3g)

Fig.3 Morphology, surface and transverse view of U. californica samples a. voucher specimen collected from Nanao Island, Shantou, China (MBM287039); b. voucher specimen collected from Pingtan, China (MBM287041); c. voucher specimen collected from Putian, China (MBM287040); d. U334-1 collected from Rongcheng Bay, Weihai, China; e. surface view of thallus; f. transverse view of thallus; g. pyrenoids. Scale bars: 5 cm in a–c, 2 cm in d, and 10 μm in e–g.
3.3 Reproduction, life cycle, and morphogenesis

A total of eight individuals, including four from northern populations and other four from southern populations, were selected for reproductive induction. After 4–6 days, it was observed that almost each surface vegetative cell had become to be a germ cell sac that contained approximately 32 germ cells (Fig. 4a), then each germ cell matured with a reddish eyespot (Fig. 4b). Indicating that U. californica germ cells can be induced by cutting thalli into fragments as well (Vesty et al., 2015). Once the germ cells were fully released (Fig. 4c), they were found to gather on the light side of the petri dish with positive phototaxis (Fig. 4d). According to the results of staining with Lugol's iodine, it was confirmed that each swimming germ cell has two flagella (Fig. 4e). After the biflagellate germ cells attached to the bottom of petri dishes, their flagella began to disappear (Fig. 4f). Then two patterns of germination from a single cell had been observed: one pattern is very rare (far below 1%) in which a germination tube developed firstly (Fig. 4g); while the other pattern is overwhelmingly dominant which depends on continuous cell divisions to form a new plantlet with uniseriate cells (Fig. 4h–k). Due to the facts that all eight individuals shared same reproductive and developmental characters, especially they produce biflagellate germ cells with positive phototaxis, they were presumed to be gametophytes all.

Fig.4 Reproduction and developent of U. californica a. germ cell sacs; b. germ cells with eyespot in germ cell sacs; c. empty cells after release of germ cells; d. positive phototaxis; e. biflagellate germ cell; f. one-cell stage; g. germination tube; h–j. multi-cell stage; k. germling. Black arrow indicates the light source. Scale bars: 10 μm in a–c and e–j, 50 μm in k.

To verify that the released germ cells were indeed gametes, the MT detection was performed with all U. californica samples. 29 PRA1 nucleotide sequences from each sample, 40 homologous sequences from germinated progeny which were produced from above eight samples as mother thalli (five progeny per sample), and 12 reference sequences from GenBank were combined to construct a ML phylogenetic tree using K2+G+I model. It was clearly shown that all 12 reference sequences were divided into two clusters, i.e., mt and mt+, based on mating types. Moreover, sequences from both 29 samples and 40 progeny were located in the mt– cluster (Fig. 5). It was worth noting that 68 of the 69 sequences were identical, and only one sequence from U417-2 differed by approximately 4.3%. These results indicated that all 29 U. californica samples collected from the coastal line of China were male gametophytes, and reproduced themselves via parthenogenesis solely.

Fig.5 Maximum likelihood (ML) phylogenetic tree based on DNA sequences of PRA1 Numbers at the nodes indicate bootstrap values. GenBank accession numbers for all reference sequences were provided. Sample ID in bold represent progeny samples. mt-: mating type minus; mt+: mating type plus.
3.4 Biogeographic and phylogeographic analysis

Using reliable sequences of U. californica for each marker, all highly similar sequences (identity ≥98% for ITS or tufA, and identity ≥99% for rbcL) were downloaded from the GenBank database. In addition, all homologous sequences which have been annotated as those of U. californica for each marker were also included to construct ML phylogenetic trees. The results showed that, after elimination of both redundant and incorrectly annotated sequences, a total of 27 ITS (Supplementary Fig.S3), 31 tufA (Supplementary Fig. S4), and 15 rbcL (Supplementary Fig.S5) sequences were identified as those of U. californica respectively. The global biogeographic distribution pattern of U. californica was analyzed by mining the geographic information associated with each conspecific sequence. According to the Supplementary Table S2, it was concluded that the convinced records of U. californica occurred in North America (USA and Canada), Europe (UK, Ireland, Germany, and Italy), Asia (Japan and Korea), and Oceania (Australia and New Zealand).

To analyze the possible geographical origin and dispersal routes of U. californica around the world, a phylogeographic analysis was conducted with all available tufA or rbcL sequences of U. californica respectively. The results were shown in Supplementary Fig.S6, all tufA sequences were divided into two clades, and only those from North American were placed in both clades. In particular, the genotype from samples in China was identical to one type of North America, and to one sample from the Atlantic coast of Europe, but different from another type of North America, as well as those from Korea, the Mediterranean or Australia. The rbcL phylogenetic tree (Supplementary Fig.S7) also showed the similar results.

4 DISCUSSION

In this study, for 29 samples from both northern and southern populations along coastal mainland China, the results of phylogenetic analysis showed that, their sequences for each marker were highly genetically identical, and clustered closely with those of U. californica collected from type locations in California, USA (Fig. 2; Supplementary Figs.S1 & S2). Further, some morphological features, such as variable overall shapes, arrangement of surface cells, and number of pyrenoids (Fig. 3), were all observed to be consistent with previous descriptions for U. californica (Tanner, 1979, 1986). In addition, the germination tube, which was reported as a unique developmental structure for U. californica (Tanner, 1986), was also detected in this study (Fig. 4g). Therefore, based on these evidences of molecular, morphological, and developmental processes, it can be determined that our samples are all U. californica. Although the occurrence of this species has been reported in China (Ding et al., 2015), that is based on only morphological records of U. angusta, a synonym of U. californica, from Taiwan, China (Okamura, 1935; Chiang, 1973). Thus, this study here reported the first record of U. californica in the mainland of China.

We believed that the origin of U. californica in the mainland of China deserves special attention. U. californica was initially only found along the Pacific coast of North America (Scagel et al., 1989; Hansen, 1997). With the emergence of its new molecular records in Europe (Hayden and Waaland, 2004), Asia (Ogawa et al., 2013; Kang et al., 2014, 2019), and Oceania (Heesch et al., 2009; Kirkendale et al., 2013), U. californica has been reported as a potential alien or non-indigenous species (NIS) in Mediterranean (Wolf et al., 2012), Japan (Kawai et al., 2007), New Zealand (Heesch et al., 2009), and Australia (Kirkendale et al., 2013). In Germany, this species is thought to be a recent introduction, but has already shown a strong ability to spread locally (Steinhagen et al., 2019). Due to the frequent occurrence of large-scale green tides in the coastal areas of mainland China in recent decades, a large number of molecular investigations have been carried out in a wide area for the risk management of green tides (Zhang et al., 2011, 2018; Duan et al., 2012; Liu et al., 2013a, b; Zhao et al., 2018; Xie et al., 2020; Cai et al., 2021). However, U. californica has never been found until our recent investigations in 2020 and 2021. Combined with the fact that southern and northern populations are highly genetically identical despite being more than 1 300 km apart, here we believed that U. californica was a recent alien species to mainland China with a rapid local spread. To analyze the possible geographical origin and dispersal routes of U. californica around the world, the phylogeographic analysis was performed with all available tufA or rbcL data (Supplementary Figs.S6 & S7). The results showed that at least two genotypes were detected in North America, with one widely distributed along the Pacific and Atlantic coasts, as well as in Texas (type I), while the other was found only in Texas (type Ⅱ) (Supplementary Fig.S6). It was indicated that all samples in China belonged to the type I, together with those records in the Atlantic coasts of Europe, while other records in Korea, the Mediterranean, Australia, maybe including that in Japan, were same as the type II (Supplementary Figs.S6 & S7). Therefore, here we hypothesized that North America was the origin of U. californica, and that the spread of this species from North America took different routes. This could be further verified by developing new high-resolution organelle-derived molecular markers (Cai et al., 2021), or even by analyzing endosymbiotic bacterial communities (Aires et al., 2013; Ghaderiardakani et al., 2020). In addition, some morphological records of U. californica in Central America (Mexico), South America (Argentina), Africa (Senegal), Middle East (Iran), and Antarctic area (Guiry and Guiry, 2022), also need to be confirmed with molecular markers.

Interestingly, this study revealed an obvious discontinuous distribution pattern of U. californica along coastal mainland China, with one part of the habitat along the North Yellow Sea and the other in the East China Sea and South China Sea. This type of pattern was first reported by Tseng and Chang (1959) in a variety of macroalgae, and proposed the following possible mechanisms. The Kuroshio flows northward through Taiwan, China, and the East China Sea, then produces small branches into the east side of Yellow Sea, which could arrive to the Shandong or Liaodong Peninsula. This movement of current was considered the main physical driving force, carrying seaweed reproductive cells for a long-distance introduction. Based on the locations of two parts of habitat revealed in this study, and the facts that almost each vegetative cell could produce germ cells when matured, we thought that the above hypothesis is also applicable to U. californica, facilitating its discontinuous distribution pattern in China. In particular, combining the observations to the reproductive process and the results of MT identification, it was indicated that all 29 U. californica samples collected in China were male gametophytes, and self-reproduced only through parthenogenesis (Figs. 4 & 5). These results implied that the northern and southern populations were not from two separate introductions, but rather from one introduction event of a single population, which was composed of male gametophytes only, and rapidly expanded through parthenogenesis. Parthenogenesis has long been reported in Ulva (Hoxmark, 1975; Phillips, 1990), and it has been proved in U. prolifera that both mother thalli and parthenogenetic progeny would share the same mating-type markers (Liu et al., 2022). Our finding provided evidences that the ability to reproduce in a variety of ways may play an important role in the spread of Ulva species. To our knowledge, this is the first study to reveal how an alien Ulva species reproduce during its population expansion in new habitats.

The introduction of NIS may pose a variety of ecological impacts on new habitats. Some studies have suggested that some green tide events, including those in the South China Sea (Xie et al., 2020), Gulf of Mexico (Melton III et al., 2016), and so on, were caused by non-native Ulva species. In recent decades, the occurrence of green tides in global oceans was obviously on the rise (Smetacek and Zingone, 2013), especially in wide coastal areas of China including the Yellow Sea (Huo et al., 2013), Bohai Sea (Song et al., 2019), and South China Sea (Xie et al., 2020). It should be noted that U. californica is also capable of being free-floating to form green tides (Bae, 2010). Therefore, early monitoring of the spread and distribution of NIS of Ulva, especially the green tide species, is of great significance for management of marine ecological risk.

5 CONCLUSION

In this study, the first record of U. californica in the mainland of China was reported, with a discontinuous distribution pattern that northern populations was in the Yellow Sea and southern populations in the East China Sea and South China Sea. It was indicated that all U. californica samples were male gametophytes, and reproduced themselves through parthenogenesis solely. Combined with the fact that southern and northern populations are highly genetically identical, here we believed that U. californica was a recent alien species to mainland China with a rapid local spread. This finding provided evidences that the ability to reproduce in a variety of ways may play an important role in the spread of Ulva species, as well as essential basic data for marine risk management of green tides in China. In addition, according to the phylogeographic analysis, the possible geographical origin and global dispersal routes of U. californica were also proposed.

6 DATA AVAILABILITY STATEMENT

The datasets analyzed during the current study are available from the corresponding author on reasonable request.

Electronic supplementary material

Supplementary material (Supplementary Tables S1–S2 and Figs.S1–S7) is available in the online version of this article at https://doi.org/10.1007/s00343-022-1392-y.

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