Tuesday 30 June 2020

Investigating the genetic diversity of Sea Squirts in Rongcheng Bay, Shandong, China.

Ascidians, or Sea Squirts, are the most abundant class of the subphylum Tunicata and are distributed along shorelines worldwide. They are sessile marine invertebrates and are widely used as a model organism for developmental and evolutionary studies. Ascidians exhibit multiple morphological characteristics, from small colonial to colorful and large solitary forms. They are divided into three major well-accepted orders, namely, Phlebobranchia, Aplousobranchia, and Stolidobranchia, based on the branchial sac morphology of the adults. However, the class Ascidiacea is paraphyletic (i.e. not everything thought to be decended from the last common ancester of the group is considered to be am Ascidian) with the  Phlebobranchia and Aplousobranchia showing a close relationship with Thaliaceae (Pyrosomes, Salps, and Doliolids), a non-Ascidian Tunicate class, whereas the Stolidobranchia remains a distinct and monophyletic group. Over the course of several decades, the Ascidiacea have been shown to be an important class of ecological species because of their invasive potential along with their ability to adapt to new environments. Transportation of Ascidians attached to ship hulls as fouling material and within the ballast water of ships has enabled them to invade new territories. This phenomenon has major impacts on local marine biodiversity as well as aquaculture industries. Therefore, the Ascidiacea were recently considered as important model species for the study of nonindigenous species worldwide.

In a paper published in the journal Ecology and Evolution on 10 March 2020, Punit Bhattachan and Runyu Qiao of the Key Laboratory of Marine Genetics and Breeding at the Ocean University of China, and Bo Dong, also of the Key Laboratory of Marine Genetics and Breeding at the Ocean University of China, and of the Laboratory for Marine Biology and Biotechnology at the Qingdao National Laboratory for Marine Science and Technology, and the Institute of Evolution and Marine Biodiversity at the Ocean University of China, present the results of a comparative analysis of three Ascidian species from northeast of China, with samples from elsewhere in the world, using the cox1 gene sequence as a genetic marker to distinguish native from invasive ascidian populations.

Bhattachan et al. collected adults of three Ascidian species, Ciona robusta, Ciona savignyi, and Styela clava, from the Rongcheng Bay area of Shandong Province, which is a part of the Yellow Sea, in northeast China. These were maintained in the laboratory in seawater tanks with aeration and constant illumination, where species were identified morphologically, and internal tissues were collected for DNA extraction and sequencing.

Collection site of the Ascidian samples (black arrow). Bhattachan et al. (2020).

The cox1 sequences from three ascidian populations at different regions of the world were retrieved from the NCBI database to build multiple sequence alignments. Only the unique haplotype datasets were used for the multiple sequence alignments. Neighbor-Joining  and maximum parsimony  methods were employed to construct a phylogenetic tree with 1000 bootstrap estimations in the default setting using MEGA7.0. The barcode region of the cox1 sequence (accession no. HM151268.1) of the Sea Pinapple, Halocynthia roretzi, was used as an out-group.

Multiple sequence alignments of cox1 from the three Ascidian species were performed separately in ClustalW hosted by MEGA7.0 using default settings. Genetic diversity parameters, including haplotype number, haplotype diversity, nucleotide difference, mutation number per sequence, number of segregating sites, and nucleotide diversity, were estimated using DnaSP software.

Relationships among the three Ascidians cox1 haplotypes found globally, including those from China, were determined using a median-joining method in the network software. To infer the population structure and understand the connectivity between native and invasive ascidian populations, we performed molecular variance analysis using cox1 haplotypes from samples available in the database as well as those in Bhattachan et al.'s dataset study using the ARLEQUIN 3.11 software.

Morphological identification of the three Ascidian species. (b) Ciona robusta adult with oral siphon (os), atrial siphon (as), sperm duct (white arrow), oviduct (black arrow), and red colour at the tip of the sperm duct (arrowhead). (c) Ciona savignyi adult with oral siphon (os), atrial siphon (as), sperm duct (white arrow), and oviduct (black arrow). (d) Adult Styela clava with oral siphon (os) and atrial siphon (as). Scale bar represents 1 cm. Bhattachan et al. (2020).

Bhattachan et al. cloned the full length of the cox1 gene from genomic DNA of 50 individuals of the three Ascidian species. Each resulting sequence was subjected to BLASTN, with the results indicating that these sequences belonged to the three respective ascidian species. The open reading frames of the cox1 sequence from three species were variable. Bhattachan et al. identified a deletion polymorphism of cox1 in Ciona savignyi, but not in Ciona robusta and Styela clava. For instance, only a single 1560 and 1543 base pair-length of cox1 sequence was identified in Ciona robusta and Styela clava, respectively, whereas two different lengths of cox1 (1545 and 1548 base pairs) were identified in Ciona savignyi. All these sequences were deposited in the NCBI database.

Bhattachan et al. also retrieved the cox1 barcode sequences from the NCBI database Only the cox1 barcode regions of unique haplotypes were used for multiple sequence alignments and phylogenetic tree construction. The resulting phylogenetic trees allowed us to delineate different haplotypes among all of the samples. In the Ciona robusta tree, Bhattachan et al. found that the haplotypes (H_1 to H_9) from China did not form a single clade in either Neighbor-Joining or maximum parsimony trees, but rather clustered with some haplotypes from individuals originating from Korea and the USA. Similarly, the haplotypes (H_1 to H_16) of Ciona savignyi from China did not cluster in a single clade in either Neighbor-Joining or maximum parsimony trees. Instead, they grouped with other haplotypes from Korea and the USA. In addition, Neighbor-Joining and maximum parsimony trees did not resolve the haplotypes (H_1 to H_14) of Styela clava from China into a single clade either. Conversely, they formed a cluster with some haplotypes from New Zealand and the USA, which were invasive populations.

Bhattachan et al. used the cox1 gene for molecular diversity analysis. Nine haplotypes were identified among 14 Ciona robusta samples, 14 haplotypes among 19 Styela clava samples, and 16 haplotypes among 17 Ciona savignyi samples. The results of the comparative analysis using different genetic diversity parameters also revealed that Ciona savignyi was diverse compared with Ciona robusta and Styela clava. The haplotype diversity was comparatively higher in Ciona savignyi (0.993 + 0.038) than that in Ciona robusta (0.912 + 0.059), and Styela clava (0.947 + 0.038). Similarly, the detected average number of nucleotide difference in Ciona savignyi (20.618) was higher than that in Ciona robusta (8.143) and Styela clava (11.550). Nucleotide diversity and average number of mutations were also relatively higher in Ciona savignyi (0.02630, 0.05061) compared with Ciona robusta (0.01094, 0.01811) and Styela clava (0.01919, 0.03097), respectively.

A Tajima neutrality test produced negative values for all three species, but these values were significant only in the Ciona savignyi population, indicating that there was an excess of low-frequency polymorphisms, and the Ciona savignyi population was expanding. However, in the Ciona robusta/Styela clava populations, the values were not statistically significant, indicating that these two species populations did not deviate from the neutral expectations. Similarly, for Fu and Li's D* statistic, negative values were observed in all three species. The values from Ciona robusta and Ciona savignyi were statistically significant, whereas those from Styela clava were not. The results from these two analytical approaches indicate that the population of Ciona savignyi is undergoing positive selection and expansion.

Bhattachan et al. divided the three Ascidian species populations into native and invasive groups, with populations located within eastern Asian countries-like China, Japan, and Korea being considered as native groups. Since these species are believed to have originated from this region while the rest of the populations from other regions were grouped as invasive populations. Network analysis revealed that there were three haplogroups (1, 2, and 3) in Ciona robusta and Ciona savignyi, respectively. No haplogroups were found for Styela clava. In the Ciona robusta network, Bhattachan et al. found native populations in haplogroup 1, and haplogroup 3 consisted of invasive populations. On the other hand, haplogroup 2 was comprised mainly of native populations, including those from China, but few haplotypes were shared from invasive populations as well. Haplogroups 1 and 2 were connected with haplogroup 3. Similarly, in the Ciona savignyi network, Bhattachan et al. found native populations in haplogroup 1, but haplogroup 2 was entirely composed of only native populations, and haplogroup 3 consisted only of invasive populations. By contrast, there were no haplogroups present in the Styela clava network, and all haplotypes from both native and invasive populations, including those from China, were connected to each other.

Bhattachan et al. also performed a hierarchical analysis of molecular variance using cox1 haplotypes from both native and invasive populations of the three Ascidian species. There was no clear structure between native and invasive populations in Ciona robusta and Ciona savignyi, but these values were not statistically significant. In addition, we recorded a negative value for Styela clava, indicating that there was no population differentiation. By contrast, among populations of Ciona robusta, Ciona savignyi, and Styela clava, there were significant variations, with the highest level of variation appearing in Ciona savignyi. Surprisingly, within these variations, the highest value was recorded for Styela clava (77.37%),  followed by Ciona savignyi (22.77%) and Ciona robusta (21.07%).

Bhattachan et al. identified three Ascidian species from Northeast China using both morphological characteristics and genetic marker analysis. The tunic of Ciona spp. is soft and semi-transparent, whereas that of Styela clava is relatively rough and opaque. Since the tunic is mainly composed of a cellulose-like material resembling that of plants, we assume that tunic composition varies among different species. In addition, Ciona spp. absorb more water, as demonstrated by dry tunic weight, and potentially as a result, this organ became semi-transparent in nature. Furthermore, Ciona robusta is comparatively larger in size than Ciona savignyi. Recently, it was also revealed that the morpho-physiological properties play an essential role in the control of size between these two Ascidians. Hence, Bhattachan et al. use these characters to distinguish between them. It is also interesting to note that there is a red coloration at the tip of the sperm duct in Ciona robusta, which is absent in Ciona savignyi. The evolutionary and functional property of this pigmentation is not yet known. Strikingly, egg morphology also varies among these three species. For instance, long follicle cells are present on the outer covering of Ciona robusta eggs, comparatively shorter follicular cells overlay Ciona savignyi eggs, and no outer follicle cells are present on Styela clava eggs. Generally, the Ascidian egg consists of two layers of follicle cells, with a vitelline coat next to the egg membrane and several test cells between them. These outer follicle cells are vacuolated and elongated and are speculated to provide buoyancy to eggs in seawater. This may help Ascidian eggs disperse by the water current and thereby be transported to distant places. Follicle cells are also the first contact of sperm entry, and it is widely known that they function to prevent self-fertilization via a chemical reaction. Long follicle cells might have enabled a higher dispersal rate of Ciona robusta. This characteristic might also inhibit more self-fertilization in comparison to Ciona savignyi and Styela clava.

The genetic marker cox1 has been widely used for identification and characterization of genetic diversity. On the basis of barcode region of the cox1 gene from these three Ascidian species as well as other available sequences in the databases, Bhattachan et al. constructed the phylogenetic trees to infer their identification, which showed that the Ciona spp. from China was closely related to native populations, mostly from Korea to Japan. This result indicates that the Ciona spp. samples collected here from China are indeed native Ascidians, and these were not introduced from other geographical areas. However, Styela clava formed a clade with invasive populations. Bhattachan et al. also found that some haplotypes from invasive populations formed a cluster with native populations. This result indicates that there was incursion of native and invasive Ascidian populations to different parts of the world. A similar phylogenetic method was used for Ascidian identification in other geographical regions as well.

Ascidians are marine organisms with a relatively high level of genetic diversity, and there exist differences in levels of genetic diversity among the Ascidians themselves. How these differing levels of genetic diversity are maintained remains unknown. Bhattachan et al.'s current analyses confirmed that these Ascidians have a high level of genetic diversity, with Ciona savignyi exhibiting a comparatively high level of genetic diversity at the molecular level. One possible explanation might be that Ciona savignyi has a large effective population size, with differing life-history traits compared to Ciona robusta and Styela clava. Of note, a previous genome-wide study also revealed that Ciona savignyi exhibited the highest level of genetic diversity. Other comparative studies on Ascidians also confirmed that they have different evolutionary rates. This could be another reason causing the different levels of genetic diversity among these three species. In addition, the neutrality tests showed that Ciona robusta and Styela clava are undergoing neutral evolution, and Ciona savignyi is experiencing population expansion and positive selection. This also explains why Ciona savignyi exhibits a higher level of genetic diversity compared with Ciona robusta and Styela clava. Given the widespread distribution of Ascidians, it is possible to exhibit high genetic diversity across populations. This kind of observation is also seen in a wide range of other organisms.

Another important characteristic feature of Ascidians is their invasive potential. Some Ascidian species are dispersed to different geographical or ecological niches because of both anthropogenic and natural causes and are considered as invasive species. Bhattachan et al. compared the global cox1 haplotypes of these three Ascidians to understand their connectivity and population genetic structure. Global haplotypes were divided into native and invasive populations. The network analysis indicated that Ciona spp. formed haplogroups with separate native and invasive populations, although some haplotypes were shared. However, in the network of Styela clava, there was no such haplogroup formation as all of its haplotypes were interconnected, suggesting extensive incursion for this species in different geographical areas. A previous global study of Styela clava also suggested its extensive incursion, in which it was categorized as invasive species. In addition, a regional study of this species indicated the multiple sources of incursions. The results of the hierarchical analysis of molecular variance of the three species of Ascidian were also consistent with the network analysis. Bhattachan et al. found a weak population genetic structure in Ciona spp. and less genetic differentiation in Styela clava populations. An occasional gene flow between native and invasive populations of Ascidians might have occurred previously, most likely via ship transport. Bhattachan et al. clearly show that the Ciona robusta and Styela clava invasive potential is attributed to the neutral genetic diversity, whereas the invasive potential of Ciona savignyi might not be due to neutral evolution, but rather by population expansion and positive selection. Previous work indicated that a neutral force plays a role in the biological invasion and subsequent structuring of a population, but equally natural selection within biological invasion was also well characterised. It is worth noting that our analysis was based on the small sample size, because of the fewer collection sites. Increase of collection sites and sample sizes could be more accurate for the population genetic evaluation, but would not change the conclusion. Bhattachan et al.'s study reveals a global relationship between native and invasive populations and has implications in understanding the invasive potential of these three species. Thus, their work provides approaches useful for risk evaluation and management of invasive species.

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