The lancelets are widely distributed in Atlantic Ocean, Indo-West Pacific Region and other waters^[1]. Gray, in 1847, first named the amphioxus from Borneo(now Sarawak state, Malaysia) Amphioxus belcheri^[2]. It was later renamed Branchiostoma belcheri which distributes along the east coast of Asiatic mainland from South China Sea to Bohai Sea and Japanese waters. There have been many discussions on the taxonomic status of amphioxus along the Pacific coast^[3-6]. Currently, differentiation of the amphioxus is so obscure that the taxonomic status of B. belcheri tsingtauense remains controversial^[7].

The recent progress in molecular biological techniques like isoenzyme technique, DNA sequencing and DNA molecular marker has made the exploration of genetic diversities of organisms more straight forward, and the genetic analyses aided by these techniques have been effective particularly in evolutionary studies of morphologically similar organisms. Indeed, comparative genetic studies of congeneric animals using molecular markers have revealed large genetic differentiation^[8], suggesting that their general body organization such as the lancelets has remained stable for a long time following phylogenetic splitting. Therefore, although the lancelets have shown few morphological changes, it is possible that they have accumulated significant genetic changes at molecular level and the diversification among the extant species can be examined by genetic survey^[9].

In the present study, two Branchiostoma species from the coast of China in Pacific Ocean were genetically surveyed by mitochondrial DNA sequence analysis to examine the extent of their genetic differentiation from the two species mentioned above.

1.   Materials and methods

1.1   Species

Four samples were collected from Shazikou in Qingdao(qd1~qd4) in 2004 and another four samples from Xiamen(xm1~xm4). All samples were fixed soon after collection and preserved in 95% ethanol until DNA extraction. Bahamas were used as an out-group in the present phylogenetic analysis. Data for B. malayanum; B. lanceolatum and B. belcheri were obtained from the Genbank, accession numbers for the species being AB105138, Y16474, AB105136, AB078191, AB083383, AB083384, AB083385, AB105136, AY700108 and AY700110, respectively.

1.2   DNA extraction, PCR and sequencing

Four amphioxus individuals each from Qingdao coded as qingdao1, qingdao2, qingdao3 and qingdao4 and four from Xiamen as xiamen1, xiamen2, xiamen3 and xiamen4 were used for DNA extraction. Total DNA of each individual was isolated from the digested tissue solution using a standard phenol-chloroform method and ethanol precipitation^[10].

The mitochondrial DNA of amphioxus was amplified with the following primers: COI_R: (5′-TAA TAG TAG TAA ATA AGC AGT-3′) and COI_F: (5′-CTA CTA ATC ATA AAG ATA TTG G-3′); Cytb-amp-R: (TGT TGC ATT ATC AAC AGA AA) and Cytb-amp-F: (AGA ATT TAA GCA TGA AAA GC)^[11]. PCR was carried out on a thermal cycler(Biometra Tgradient) for 38 cycles with the following parameters: preheating at 95℃ for 2 min, denaturation at 94℃ for 45 sec, annealing at 48℃ for 45 sec, and extension at 72℃ for 45 sec. After the final cycle, an extra extension at 72℃ continued for 5 min, and then, the PCR product was held at 4℃. Following the examination of PCR products with agarose gel electrophoresis, amplified DNA fragments were purified with Wizard PCR Preps DNA Purification System(Promega). Purified double-strand DNA was directly sequenced using the two PCR primers from both directions on a CEQ 8000 Genetic Analyzer(Beckman Coulter).

1.3   Sequence analysis

Raw data of each sequence was reevaluated using DNASTAR package. Then the obtained sequence alignment was initiated with Clustal X^[12], subsequent inspection and correction being made manually. Genetic relationships were inferred using the UPGMA and the neighbor-joining (NJ) method^[13] with Kimura′stwo-parameter model implemented in Mega Ver.2.0^[14].Mega was also used for calculation of nucleotide sequence differences(p-distances). The robustness of each internal branch of the phylogenetic trees was evaluated with 1 000 bootstrap replicates^[15].

2.   Results

2.1   Sequence divergence among the amphioxus

We used the Clustal X to deal with these sequences getting a 520 bp and 518 bp DNA fragment of COI and Cyt b gene, respectively. The average of nucleotide compositions of four Qingdao sample based on COI gene were 38.1%, 15.2%, 22.7%, 24.0% for T, C, A and G, respectively, and those of four samples from Xiamen waters average were 39.4%, 15.3%, 22.0%, 23.3% for T, C, A and G, respectively. The values based on the Cyt b gene were 40.5%, 15.2%, 21.9%, 22.5% for T, C, A and G, respectively in Qingdao specimens, and 40.9%, 13.9%, 22.9%, 22.4%, respectively in Xiamen specimens. After homologous comparison of Branchiostoma species from Qingdao and Xiamen waters by Cyt b gene, we found three haplotypes in Qingdao samples and two in Xiamen samples which showed the genetic diversity in Qingdao and Xiamen amphioxus population, respectively(Fig. 1).

Figure  1.  Variable sites in a 518 bp fragment of Cyt b gene of amphioxus

下载: 全尺寸图片 幻灯片

The percentages of pairwise difference are listed in Tab. 1 and Tab. 2. The percentages of pairwise difference within Qingdao amphioxus based on COI gene average 0.42%(Tab. 1), and those within four samples from Xiamen average 0.23%. Surprisingly, the genetic distance between Qingdao amphioxus and Japanese amphioxus averages only 1.3%, but that between Xiamen and Japanese amphioxus(19%) is very similar to the distance between Qingdao and Xiamen amphioxus(18.8%). The same result can be found according to the Cyt b gene. The percentages of pairwise distances were only 1.4% within Qingdao specimens and 0.4% in Xiamen specimens. The values between Xiamen and Japanese amphioxus(19.9%) were very similar to that between Qingdao and Xiamen amphioxus(20.3%) determined by the Cyt b gene, and the genetic distance between Qingdao and Japanese amphioxus average only 0.9%(Tab. 2).

Table  1.  Percentage of pairwise differernce in COI gene of lancelets(below diagonal) and number of transitiongs/transversions(above diagonal)

	xm1	xm2	xm3	xm4	qd1	qd2	qd3	qd4	jap	mala	lan	luc
xm1		0/0	0/1	1/1	47/29	47/29	48/29	47/29	48/29	42/32	49/34	49/45
xm2	0.000		0/1	1/1	47/29	47/29	48/29	47/29	48/29	42/32	49/34	49/45
xm3	0.002	0.002		1/0	47/28	47/28	48/29	47/28	48/28	42/31	49/35	50/44
xm4	0.004	0.004	0.002		47/28	47/28	48/29	47/28	48/28	42/31	48/35	51/44
qd1	0.188	0.188	0.186	0.186		0/0	2/0	2/0	5/0	43/27	46/31	51/44
qd2	0.188	0.188	0.186	0.186	0.000		2/0	2/0	5/0	43/27	46/31	51/44
qd3	0.191	0.191	0.189	0.189	0.004	0.004		4/0	7/0	43/27	46/31	53/44
qd4	0.188	0.188	0.186	0.186	0.004	0.004	0.009		7/0	41/27	44/31	49/44
jap	0.191	0.191	0.189	0.189	0.011	0.011	0.015	0.015		44/27	47/31	51/44
mala	0.182	0.182	0.179	0.179	0.171	0.171	0.171	0.166	0.174		52/32	58/45
lan	0.208	0.208	0.211	0.208	0.191	0.191	0.191	0.185	0.194	0.212		58/43
luc	0.239	0.239	0.240	0.243	0.243	0.243	0.249	0.236	0.243	0.268	0.262
Note: xm. Xiamen amphioxus; qd. Qingdao amphioxus; jap. Japanese amphioxus; mala. B. malayanum; lan. B. lanceolatum; luc. Epigonichthys lucayanus.

下载: 导出CSV 

| 显示表格

Table  2.  Percentage of pairwise differernce in Cyt b gene of lancelets(below diagonal) and number of transitiongs/transversions(above diagonal)

	jap3	xm1	jap1	qd2	jap4	jap2	qd1	hp	lan	xm2	qd3
jap3		48/40	6/0	6/0	5/0	3/1	7/0	79/99	60/49	48/41	3/0
xm1	0.191		48/40	51/40	49/40	49/41	50/40	80/105	55/49	1/1	49/40
jap1	0.010	0.194		6/0	5/0	3/1	7/0	81/99	58/49	48/41	3/0
qd2	0.012	0.199	0.010		7/0	3/1	7/0	80/99	60/49	51/41	3/0
jap4	0.008	0.196	0.010	0.012		4/1	8/0	80/99	59/49	49/41	4/0
jap2	0.008	0.196	0.006	0.008	0.008		4/1	79/100	59/50	49/42	0/1
qd1	0.014	0.196	0.012	0.014	0.014	0.010		81/99	59/49	50/41	4/0
hp	0.477	0.508	0.485	0.481	0.481	0.481	0.485		97/113	82/106	79/99
lan	0.251	0.236	0.245	0.251	0.248	0.251	0.248	0.613		55/50	59/49
xm2	0.194	0.004	0.196	0.202	0.199	0.199	0.199	0.516	0.239		49/41
qd3	0.006	0.194	0.004	0.006	0.006	0.002	0.008	0.477	0.248	0.196
Note: jap. Japanese amphioxus; xm. Xiamen amphioxus; qd. Qingdao amphioxus; hp. Ciona intestinalis; lan. B. lanceolatum

下载: 导出CSV 

| 显示表格

Besides the percentage of nucleotide differences, homologous comparison also indicates that transversions seldom occur among individuals within the population^[16]. It is so between Qingdao and Japanese amphioxus, but 28/29(Tab. 1) and 40/41(Tab. 2) transversions were present between Qingdao and Xiamen amphioxus based on COI and Cyt b gene, respectively. The same result can be found between Japanese and Xiamen amphioxus(28/29 and 41/42). The amphioxus from Qingdao and Japanese waters differed significantly from the amphioxus of Xiamen waters.

2.2   Phylogenetic relationships

Two phylogenetic trees showed that the Qingdao and Xiamen species pairs were reciprocally clustered into two significantly different clades, supported by high bootstrap values(Fig. 2).The phylogenetic trees based on the Cyt b gene(Fig. 3) showed the same result with the phylogenetic trees based on the COI gene, supporting that the Qingdao and Xiamen species pairs belong two significantly different clades and the amphioxus from Qingdao and Japanese belong to the same clade. The two results strongly supported that Qingdao and Japanese lancelets, Xiamen lancelets and lancelet like B. lanceolatum from Atlantic Ocean belong to the different clades, and have far genetic distance reciprocal.

Figure  2.  UPGMA phylogenetic tree for amphioxus based on partial COI gene sequence

下载: 全尺寸图片 幻灯片

Figure  3.  NJ phylogenetic tree for amphioxus based on partial Cyt b gene sequence

下载: 全尺寸图片 幻灯片

3.   Discussion

In 1936, Tchang and Koo reported the new distribution of Chinese amphioxus in the Kiaochow Bay(southern part of the Shandong peninsula) and named it as a new variety B. belcheri tsingtauense on the basis of its morphological differentiation. From that time on, a commonly accepted opinion is that the lancelet along the east coast of China from South China Sea to Bohai Sea and Japanese waters is B. belcheri as first named by Gray in 1847. But the survey using the morphological method and DNA molecular techniques appears to show that the differentiation has occured among the amphioxus along the coast of China and the taxoniomic status of B. belcheri tsingtauense has remained controversial. According to the Tab. 1, the genetic distance between amphioxus mentioned above in the two places determined from COI gene is 18.8% on average, which is the same as that observed in other interspecific variations^[9]; and the genetic distance determined by partial Cyt b gene(Tab. 2) between amphioxus of the two places is 20.3% which is much higher than that observed in other intraspefic variations(usually < 5%)^[17]. The result of genetic distance is coincident with the phylogenetic trees reconstructed by COI and Cyt b gene, obviously showing that the amphioxus from Qingdao and Xiamen in the different clades(Fig. 2, Fig. 3). Besides the percentage of nucleotide differences between Qingdao and Xiamen amphioxus, comparing specimens from Qingdao and Xiamen with Epigonichthys lucayanus as the outgroup in the pairwise difference, the genetic distance are near to 24% and 23%, respectively(Tab. 1, Tab. 2). From the genetic point of view, the distance between Qingdao and Xiamen amphioxus appears to be closely related to that between Qingdao amphioxus and E. lucayanus, also Xiamen amphioxus and E. lucayanus. All this genetic information strongly indicate that the amphioxus from Qingdao and Xiamen should belong to different species; we support the viewpoint that the original subspeciences B. belcheri tsingtauense should be elevated to species level on the nomenclatorial scale, namely B. tsingtauense. Geographic events may have played some roles in bringing about such genetic differentiation(e.g. glacial period).

According to the present survey including the morphological research^{[5, 18]} and molecular surveys, we suggest that amphioxus in East China might belong to different species and we utmostly support the viewpoint that the original subspecies B. belcheri tsingtauense should be renamed as a separate species and its new scientific name should be B. tsingtauense which includes amphioxus both in Bohai Sea and in Japanese waters. Accordingly, amphioxus in Xiamen, including those distributed in this area, should keep the name of B. belcheri.