Taxonomic status and phylogeographical structure in Pseudonereis sp. East Asia in the East China Sea off the Coast of Zhejiang, China

doi:10.21203/rs.3.rs-3291494/v1

Download PDF

Research Article

Taxonomic status and phylogeographical structure in Pseudonereis sp. East Asia in the East China Sea off the Coast of Zhejiang, China

https://doi.org/10.21203/rs.3.rs-3291494/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Pseudonereis variegata is a polychaeteous worm (Annelida: Nereididae) with a cosmopolitan distribution in coastal waters; however, noticeable variations in morphology and genetics raised the question whether individuals from distinct ocean basins belong to the same species. This study evaluated the taxonomic status and phylogeographic structure in Pseudonereis sp. East Asia (as P. variegata) in the East China Sea (ECS) off the Coast of Nanji Islands (NJ) and Zhoushan Islands (ZS), Zhejiang by analyzing a 582bp DNA sequence in mitochondrial cytochrome C oxidase subunit I (COI). A total of 36 haplotypes were scored from 152 specimens, including 40 from ZS and 112 from NJ. Between NJ and ZS, 5 haplotypes were in common. Relatively, the levels of diversity on NJ (Hd = 0.556±0.060, π = 0.149±0.023%) were higher than ZS (Hd = 0.478±0.099, π = 0.110±0.028%). Comparative analyses with morphological characters and COI sequences of congeneric samples derived from other locations indicated that Pseudonereis sp. East Asia in the West Pacific, including the East China Sea/Taiwan Strait (ECS-TS) and Yellow Sea (YS) had a strong and significant divergence from the conspecific specimens in Chile and thus they should be described as a separate species, namely Pseudonereis sp. East Asia. The lack of noticeable phylogeographic structuring observed among populations in Pseudonereis sp. East Asia is probably due to larval transportation and oceanographic connection via the coastal currents. Our study provides basic scientific information for understanding the identity and genetic diversity of Pseudonereis sp. East Asia in the East China Sea off the Coast of Zhejiang, China and neighboring areas.

Pseudonereis variegata

Species delineation

Nanji Islands

Zhoushan Islands

COI

Genetic diversity

taxonomic status

phylogeography

Polychaeta is the largest class in the phylum, Annelida, including over 80 families, 1,600 genera, and more than 20,000 described species (Fauchald 1977; Pettibone 1982; Hutchings & Murray 1984; George & Hartmann-Schröder 1985; Yang & Sun 1988). These animals have a widespread distribution in geographically distant ocean basins and play important roles in energy flow and nutrient cycling in benthic ecosystems (Neira & Höpner 1994; Beesley et al. 2000; Barnes et al. 2009). It is generally believed that the dominant manner of dispersal (Jackson 1986) in sessile or sedentary benthos is through pelagic larvae (Scheltema 1968, 1971, 1988). However, some cosmopolitan species living in littoral sediments seem to lack pelagic stages (Giere 2008). Recently, a growing number of studies reported the occurrence of cryptic species complexes. As a consequence, cosmopolitan distributions should be taken with caution (Schmidt & Westheide 2000; Barroso et al. 2010; Nygren 2014; Silva et al. 2017; Hutchings & Kupriyanova 2018). About 1,000 polychaete species in 60 families have been reported from Chinese coastal waters (Ge et al. 2018), including 74 nereidid species (Wu et al. 1985; Sun & Yang 2004).

The nereidid or sandworm (Pseudonereis variegata Grube 1857) was first described as Nereilepas variegata (Grube 1857) from Chile and Peru. It is an intertidal species living in the beds of mussels, tube worms and barnacles, and exploited as food or bait in some areas (Van Herwerden 1989). P. variegata used to be considered a circumtropical species, widely distributed in the Grand Caribbean region, South America, Africa, South Pacific Islands, Indian coastal waters, and East Asia (Gibbs 1969, 1972; Wu et al. 1985; Van Herwerden 1989; Bakken 2007; Satheeshkumar & Jagadeesan 2010; Conde-Vela 2018; A Abdelnaby 2020). In general, nereidids are dioecious and semelparous. Adults of many nereidids, including P. variegata (Sato 2017), conducted external fertilization in water, including a metamorphosis process, from epitokous to typical heteronereis form.

Noticeable variations in morphology and genetics raised the question whether individuals from distinct ocean basins belong to the same species (Tosuji & Sato 2010; Sampertegui & Rozbaczylo Narváez 2013; Paiva et al. 2019; Kara et al. 2020). So far, there is compelling evidence for the occurrence of cryptic species in P. variegata, e.g. Nereis ferox (Hansen) recently described from Brazil and P. podocirra (Schmarda) from South Africa differed from P. variegata from Chile (Conde-Vela 2018; Kara et al. 2018). However, many polychaete species lack genetic data or integrative taxonomic revisionary information combining key morphological and molecular data (e.g. ultrastructure investigation (Knowlton 1993, 2000). Because of the shortage of experts in polychaete taxonomy and lack of identification keys, it often results in unreliable species identifications. Reports of specimens collected far from the species’ type locality should be cautioned (Hutchings & Kupriyanova 2018), prior to accurate morphology-based identification (Lobo et al. 2016).

Zhoushan Islands (ZS) and Nanji Islands (NJ) lie in the East China Sea (ECS) off the Coast of Zhejiang Province, China. On the northern Coast of Zhejiang, ZS is located outside the Yangtze River and Hangzhou Bay and has strong impacts by freshwater runoffs from the Yangtze, Qianjiang and Yongjiang River, which supports a wide variety of fisheries species by providing spawning and feeding habitats (Liu et al. 1991); on the southern Coast of Zhejiang, NJ is located outside the Aojiang River Estuary. It is one of the earliest Marine Nature Reserves established in China, hosting a high level of marine biodiversity particularly shellfish and algae (Tang et al. 2014). NJ also provides a step stone and critical habitat that facilitates numerous marine species to disperse and migrate via the Taiwan Warm Current and Jiangsu and Zhejiang Coastal Currents along northern latitudes (Li et al. 2009).

DNA sequences are widely used in identification and delineation of species that lack diagnostic morphological characters (Abdel-Mawgood 2012). Mitochondrial cytochrome C oxidase subunit I (COI) has an evolutionary rate suitable in characterizing variations among geographical populations of a species and among closely-related species in many groups of marine organisms (Daniels et al. 2002; Hebert et al. 2003; Tornabene et al. 2010), including polychaetes (Sampertegui & Rozbaczylo Narváez 2013; Kara et al. 2018; Paiva et al. 2019). COI can complement traditional morphological identification for detection of cryptic species and population genetic structure. Using COI DNA sequences, this study aimed to determine: (1) whether P. variegata specimens collected from ECS off the Coast of Zhejiang are the same species as P. variegata from the type locality in Chile and other geographically distant ocean basins, and (2) the genetic diversity, phylogeographical structure and demography history in P. variegata in ECS and other geographically approximate areas.

Sample collection and morphological identification

Specimens were collected from seven locations on ZS: Qushan Island-Shalong, (QS), Qingbang Island-Mazuitou (QB), Xifushan Island (XFS), Tangtou (TT), Zhujiajian Island-Xiaowushitang (ZJJ), Xiazhi Island-Henicao (XZ), Dawenchong Island (DWC), and ten sites on NJ: Guoxingao (GXA), Houjishan Island (HJS), Mazuao (MZA), Sanpanwei (SPW), Sanjiaoliao (SJL), Longchuanjiao (LCJ), Shangmaan Island (SMA), Xiamaan Island (XMA), Dachaiyu Island (DCY) between May 2015 and November 2019. These two archipelagos lie in ECS off the northern and southern Coast of Zhejiang Province, China (see map on Fig. 1 and Table 1 for sampling details).

Table 1

Species, GenBank accession number, putative species group (PSG), geographical origin, and source of reference *COI* sequence in *Pseudonereis* spp used in this study.
Species	GenBank Accession #	PSG	Geographical origins	Reference source
Pseudonereis sp. East Asia	OR413633-OR413648	1	ZS, Zhejiang	This Study
Pseudonereis sp. East Asia	OR413649-OR413691	1	NJ, Zhejiang	This Study
Pseudonereis sp. East Asia	JX503027	1	South Korea	Kim, S. et al.
Pseudonereis sp. East Asia	JX503028	1	South Korea	Kim, S. et al.
Pseudonereis sp. East Asia	JX503029	1	South Korea	Kim, S. et al.
Pseudonereis sp. East Asia	KC800617	1	Qingdao, Shandong	Deng, Y
Pseudonereis sp. East Asia	KC800618	1	Qingdao, Shandong	Deng, Y
Pseudonereis sp. East Asia	KC800619	1	Qingdao, Shandong	Deng, Y
Pseudonereis sp. East Asia	KC800620	1	Qingdao, Shandong	Deng, Y
Pseudonereis sp. East Asia	KC800621	1	Qingdao, Shandong	Deng, Y
Pseudonereis sp. East Asia	KC800622	1	Qingdao, Shandong	Deng, Y
Pseudonereis sp. East Asia	KY129878	1	Nanji, Zhejiang	Chen, Y. et al.
Pseudonereis sp. East Asia	KY129879	1	Xiapu, Fujian	Chen, Y. et al.
Pseudonereis sp. East Asia	KY129880	1	Cangnan, Zhejiang	Chen, Y. et al.
Pseudonereis sp. East Asia	KY129881	1	Dongtou, Zhejiang	Chen, Y. et al.
Pseudonereis sp. East Asia	MN256547	1	Putian, Fujian	Xing, B. P. et al.
Pseudonereis sp. East Asia	MN256548	1	Putian, Fujian	Xing, B. P. et al.
Pseudonereis sp. East Asia	MN256549	1	Xiapu, Fujian	Xing, B. P. et al.
P. podocirra	MH766302	2	South Africa	Jyothi Kara et al.
P. podocirra	MH766306	2	South Africa	Jyothi Kara et al.
P. podocirra	MH766319	2	South Africa	Jyothi Kara et al.
P. podocirra	MH766320	2	South Africa	Jyothi Kara et al.
P. pseudonoodti	JF293312	3	Colombia	Hererra, L. et al.
P. pseudonoodti	JF293313	3	Colombia	Hererra, L. et al.
P. anomala	JX420277	4	Australia	Glasby, C. J. et al.
P. anomala	JX420276	4	Australia	Glasby, C. J. et al.
P. gallapagensis	JF293310	5	Colombia	Hererra, L. et al.
P. gallapagensis	JF293309	5	Colombia	Hererra, L. et al.
P. variegata	HQ705183	6	Chile	Sampertegui, S. et al.
P. variegata	HQ705197	6	Chile	Sampertegui, S. et al.
P. variegata	HQ705198	6	Chile	Sampertegui, S. et al.
P. variegata	HQ705199	7	Chile	Sampertegui, S. et al.

Specimens were collected from locations in sandy, rocky and/or muddy intertidal zones. All specimens were preserved in 95% ethanol at the point of collection and later stored at -4°C for morphological and molecular analysis. Some of the specimens were anaesthetized using 7% MgCl₂ aqueous solution and photographed before ethanol preservation. Morphological identification followed the classification system and retrieval table of Fauchald (1977) modified by Yang and Sun (1988) and Sun and Yang (2004), in addition to Pettibone (1986) adopted by Sun and Yang (2004).

DNA extraction, PCR, and DNA sequencing

The extraction of genomic DNA from tissue samples followed the salt-extraction protocol (Aljanabi and Martinez 1997). PCR of COI sequence was conducted using the pair of primers in (Folmer et al. 1994). A 25 µL reaction volume included 12.5 µL of Taq Master Mix (New England Bio Labs), 9.5 µL ddH₂O, 0.5 µL of forward and reverse primer at 10 mM concentration, 1 µL of 1% bovine serum albumin and 1 µL of template DNA. Thermal cycling conditions included an initial denaturation of 95°C for 3 min, followed by 35 cycles of 94°C for 30 s, 45°C for 30 s, 72°C for 1 min, with a final extension of 72°C for 7 min. Quality PCR amplicons were sequenced at Sangon Biotech (Shanghai, China) Co., Ltd. (http://www.sangon.com) using the ABI Genetic Analysis System.

Data analysis

DNA sequences were manually assembled and corrected in Sequencher v.5.4 (Gen Codes), aligned in ClustalX v.2.1 (Larkin et al. 2007), and then edited and trimmed in BioEdit v.7.0.9 (Hall 1999). The sequences of specimens were compared using BLAST algorithm (Zhang et al. 2000) on GenBank for preliminary species identification and sequences matching. Haplotype data for subsequent analysis were generated in DnaSP v. 5.10 (Librado & Rozas 2009). Indices of genetic diversity were estimated, including the number of haplotypes (h), variable sites (S), haplotype diversity (Hd) and nucleotide diversity (π) in DnaSP v.5.10.

GTR + I + G mode was selected as the best-fit substitution model under Akaike information criterion (AIC) in jModelTest (Darriba et al. 2012). Maximum likelihood (ML) analysis in 1,000 bootstrap resampling replicates was performed in MEGA v.10.0.5 (Kumar et al. 2018). Bayesian inference (BI) of trees used MrBayes 3.1.2 (Ronquist & Huelsenbeck 2003) in 1×10⁷ generations of Markov-Monte Carlo Chains (MCMC) and four chains each run, being sampled each 1000 generations with a burn-in of 25%. Convergence between runs (stationarity of parameters) used Tracer v. 1.7 (Rambaut et al. 2018). The analysis of automatic barcode gap discovery (ABGD) was conducted on the online webserver https://bioinfo.mnhn.fr/abi/public/abgd (Puillandre et al. 2012), using the relative gap width (X) of 1.5 and Kimura 2-parameter (K2P) model for 20 steps. P_min chose 0.001 and P_max chose 0.2 to avoid detecting putative species groups (PSG) due to inner-structure. A binary rooted Bayesian tree was used as input for multi-rate Poisson tree processes (mPTP) analysis (Zhang et al. 2013). Inter- and intraspecific K2P distances were estimated in MEGA v.10.0.5.

A mantel test (Mantel 1967) was performed using GenAlEx (Peakall & Smouse 2006) to investigate the correlation between geographic and genetic distances. Among the sampling sites, pairwise geographical distances were calculated using script, and mean pairwise differences between populations (PiXY) were calculated in Arlequin v.3.11 (Excoffier et al. 2005). Subsequently, a TCS haplotype network (Clement et al. 2002) was constructed in PopArt v1.7 (Leigh & Bryant 2015) to visualize spatial distribution patterns and evolutionary relationships among haplotypes.

Tajima’s D (Tajima 1989) and Fu’s F_S (Fu 1997) were calculated in Arlequin v.3.11 to detect recent population expansion assuming the neutrality and mutation-drift equilibrium of COI DNA sequence. Mismatch distribution and demographic history were analyzed in DnaSP v. 5.10 (Librado & Rozas 2009). To examine demographical changes over time, a Bayesian Skyline Plot (Heled & Drummond 2008) was constructed using HKY substitution model in BEAST v2.6.3(Bouckaert et al. 2019). Two substitution rates of 3.5% and 4.7% per million years were employed to calibrate COI divergence times in Bering Strait polychaetes (Loeza-Quintana et al. 2019). Full runs used an MCMC of 5 × 10⁸ steps that logged at an interval of 10,000 steps. Parameter convergence used Tracer v1.7.2 (Rambaut et al. 2018).

Morphology

The major diagnostic characters in P. variegata specimens from China (Pseudonereis sp. East Asia; Fig. 2) are the absence of heterogomph spinigers (HS) and homogomph spinigers in anterior and posterior parapodia and neurochaetae, distinguishing them from P. variegata specimens collected from Chile and Brazil. Detailed morphological descriptions are as follows.

The anterior lobes of the mouth are pear-shaped. The two pairs of round eyes are arranged in an inverted trapezoidal or rectangular shape at the middle and posterior parts of the anterior lobes. The tentacles are short, basal segments of antennae are large, and terminal segments are spherical. There are 4 pairs of tentacles, the longest one extending posteriorly up to the node of 5th setae. The snout is large. The paragnath cones exist in areas on pharynx except Area Ⅵ: a shield-shaped bar; Area Ⅰ: 1; Area Ⅱ: 16–20 and triangular pile; Area Ⅲ: 24–30 and three crowded neat comb-rows; Area Ⅳ: 36–48 and several curved rows; Area Ⅴ: 1; Areas Ⅶ and Ⅷ: about 38 and irregularly in 3–4 rows. The large jaw at the snout has 4–6 lateral teeth. Notochaetae have only homogomph spinigers. Neurochaetae in anterior and middle polypide have compound homogomph spinigers (HS) and heterogomph falcigerous (HeF), while in posterior polypide have compound heterogomph spinigers (HeS) and heterogomph falcigers. The dorsal notopodial ligule extends rectanglely at the node of the 50th setae. There are colored glands at the dorsal brim and anterior venter and dorsal cirri on the tip.

Table 2

Comparison of diagnostic characters among *Pseudonereis variegata* from China, Chile and Rio de Janeiro, Brazil
	P. variegata	P. variegate	P. variegata	P. variegata	P. variegata
Reference/museum ID	Yang & Sun 1988	Sun & Yang 2004	Rozbaczylo & Bolados 1980	Sampertegui et al. 2013	Bakken 2007
Locality	China (intertidal)	China (intertidal)	Chile	Chile	Brazil
Length (mm)	90	90	51	42–43	26–46
Number of segments	-130	-130	130	61–123	64–94
Number of paragnaths
Area Ⅰ	1	1	1	1–2	1–2
Area Ⅱ	16–20	16–20	23–26	13–33	13–34
Area Ⅲ	24–30	24–30	72	61–87	59–76
Area Ⅳ	36–48	36–48	-	56–90	63–87
Area Ⅴ	1	1	1	1	1
Area Ⅵ	1 shield-shaped bar	1 shield-shaped bar	1 shield-shaped bar	1 shield-shaped bar	1 shield-shaped bar
Area Ⅶ-Ⅷ	38	-	39–41	37–46	18–21
Anterior parapodia
Notochaetae	HS only	HS only	HS only	HS only	HS only
Neurochaetae	HS and HeF	HS and HeF	HS, HeF and HeS	HS, HeF and HeS	HS, HeF and HeS
Posterior parapodia
Notochaetae	HS only	HS only	HS only	HS only	HS only
Neurochaetae	HeF and HeS	HeF and HeS	HS and HeF	HS, HeF and HeS	HS, HeF and HeS
Notopodial ligule	Expanded and elongate	Expanded and elongate	Expanded and elongate	Expanded	Expanded and elongate
Dorsal cirrus attachment	Sub-terminal	Sub-terminal	Sub-terminal	Sub-terminal	Terminal

Sequence polymorphisms

An alignment with a consensus length of 582 bp COI DNA sequences yielded a dataset of 152 specimens from the East China Sea (ECS) off the Coast of ZS (n = 40) and NJ (n = 112). A total of 34 variable sites and 36 distinct haplotypes were scored. Of the haplotypes, 11 were derived from ZS and 30 from NJ, with 5 haplotypes shared between ZS and NJ. The haplotype sequences were deposited in GenBank (accessions: OR413633- OR413691) and detailed information for specimens and sequences are shown in Table 1 and Fig. 1. Haplotype diversity (Hd) and nucleotide diversity (π) were 0.478±0.099 and 0.110±0.028% in ZS and 0.528±0.059 and 0.139±0.000% in NJ. Relatively, the levels of diversity on NJ were higher than ZS.

Additional 16 COI sequences retrieved from GenBank database that shared high similarity (> 90%) with the sequences of 36 haplotypes from this study were included in subsequent analyses (see the sequence sources in Table 1). In the dataset of all 168 COI sequences, a total of 42 distinct haplotypes and 38 variable sites were scored, 13 of which were parsimony-informative.

Phylogenetic and delimitation analysis

Bayesian and ML trees exhibited identical major topologies except for minor disparities. On the trees, the 49 distinct COI haplotypes were clearly classified into seven monophyletic clades. The two phylogenetic trees had no topological discrepancy and strong statistical supports on major branch nodes. The ABGD (p < 0.05) and mPTP analyses were in concordance with the classification of seven clades (Fig. 3), thus defined seven PSGs in Pseudonereis. All samples (42 haplotypes) derived from the ECS-TS off the Coast of Zhejiang and Fujian, YS off the Coast of Shandong and South Korea composing PSG1, and were named Pseudonereis sp. East Asia temporarily; PSG2 included four sequences (MH766302, MH766306, MH766319 and MH766320) in P. podocirra from South Africa; PSG3 included two sequences (JF293312 and JF293313) in P. pseudonoodti from Colombia; PSG4 included two sequences (JX420276 and JX420277) in P. anomala from Australia; PSG5 included two sequences (JF293309 and JF293310) in P. gallapagensis from Colombia; finally, PSG6 and PSG7 consisted of three sequences (HQ705183, HQ705197 and HQ705198) and one sequence (HQ705199) described in P. variegata from the type locality, Chile, respectively.

On average, the K2P distance between Pseudonereis sp. East Asia (PSG1) and PSG6/PSG7 in Chile was 22.0% (Table 3). PSG1 almost equally differed from PSG6 (21.7%) and PSG7 (21.8%). All remaining K2P distances among the different PSGs fell in the range between 18.9% (PSG2 vs. PSG5) and 24.4% (PSG4 vs. PSG7).

Table 3

K2P distances estimated from *COI* DNA sequence variation. Below and above the diagonal are K2P distances and standard error (SE) among PSGs, respectively; along the diagonal are K2P distances (SE) within PSGs, i.e. *Pseudonereis* sp. East Asia_PSG1 (West Pacific/East Asia), *P. podocirra*_PSG2 (South Africa), *P. pseudonoodti*_PSG3 (Colombia), *P. anomala*_PSG4 (Australia), *P. gallapagensis*_PSG5 (Colombia), *P. variegata*_PSG6 (Chile) and *P. variegata*_PSG7 (Chile; HQ705199)
	Pseudonereis sp. East Asia_PSG1 (East Asia)	P. podocirra_PSG2 (South Africa)	P. pseudonoodti_PSG3 (Colombia)	P. anomala_PSG4 (Australia)	P. gallapagensis_PSG5 (Colombia)	P. variegata_PSG6 (Chile)	P. variegata_PSG7 (Chile)
Pseudonereis sp. East Asia_PSG1 (East Asia)	0.002 ± 0.000	0.022	0.023	0.024	0.023	0.022	0.022
P. podocirra_PSG2 (South Africa)	0.208	0.006 ± 0.002	0.021	0.023	0.020	0.021	0.021
P. pseudonoodti_PSG3 (Colombia)	0.217	0.196	0.008 ± 0.004	0.021	0.020	0.020	0.022
P. anomala_PSG4 (Australia)	0.242	0.232	0.188	0.000 ± 0.000	0.021	0.023	0.023
P. gallapagensis_PSG5 (Colombia)	0.231	0.189	0.200	0.211	0.038 ± 0.009	0.021	0.022
P. variegata_PSG6 (Chile)	0.217	0.203	0.198	0.230	0.197	0.001 ± 0.001	0.023
P. variegata_PSG7 (Chile)	0.218	0.209	0.224	0.244	0.217	0.220	na

Haplotype network and demography history

The TCS network displayed a central dominant haplotype (n = 110; 65.5% of all samples) with a large proportion of private haplotypes (81.0%). The dominant haplotype connected with others in a varying number of mutant steps (1–4). The maximum number of mutation steps connecting a pair of haplotypes on the network was 7 and haplotype distribution appeared to be random in terms of sample geographical origins (Fig. 3).

The mantel test on Pseudonereis sp. East Asia_PSG1 showed no significant interdependency between the geographical and genetic distances for the samples from ZS and NJ (ZS: r = 0.003, p = 0.410, NJ: r = 0.018, p = 0.420, Fig. 4).

Tajima’s D and Fu’s F_S both deviated from the neutrality significantly (p < 0.05) in ZS, NJ and the entire sample of Pseudonereis sp. East Asia_PSG1, suggested that the populations probably experienced expansion in the recent history.

Under an assumption of population constant scenario, the curves of observed values fit those of expected ones, suggesting the stability in the ZS, NJ and entire PSG1 population in East Asia in the recent history (Fig. 5). Bayesian Skyline Plots generated for the entire PSG1 in East Asia revealed a tendency of demographic growth followed by the stability in population size. The tendency of population expansion was estimated to have persisted in the period from 15,000 to 10,000 years ago with a range of population effective size between 563 and 21,498 (Fig. 6).

Taxonomic status of Pseudonereis sp. East Asia

Specimens collected from China by Yang & Sun (1998) and Sun & Yang (2014) were identified as a single species of P. variegata. Despite morphological variability in parapodia, the arrangement of paragnaths in the species is similar. P. variegata specimens from China differ from the ones from the type location of Chile (collected by Sampertegui) on the shape of notopodial ligule (Sampertegui & Rozbaczylo Narváez 2013); there are also some differences in the shape of notopodial ligule and dorsal cirrus attachment between specimens from Chile and from Brazil (Rozbaczylo & Bolados 1980; Bakken 2007; Sampertegui & Rozbaczylo Narváez 2013). If these morphological diagnostic features are interspecific, the specimens from China, Chile and Brazil should be recognized as different species, although this hypothesis need be further confirmed with molecular evidence.

Comparative analyses using COI sequences indicated that Pseudonereis sp. East Asia had a strong and significant divergence from the specimens of P. variegata in Chile and other species in Pseudonereis. Thus, they should be described as a separate species. In fact, most of the seven PSGs recognized in the phylogeny by delimitation analysis distributed in separate, geographically distant ocean basins likely belong to distinct species. The interspecific genetic differences ranged from 3.7–27% in nereidids. Despite no common fixed range of value set up in species delimitation (Brett 2006; Kvist 2016), the seven PGSs in Pseudonereis had pairwise K-2-P distances between 20% and 25%, in concordance with the earlier reports (Kara et al. 2018). In addition, the two P. variegata PSGs in Chile that had a significant divergence should be recognized as distinct species and deserved further investigation (Kara et al. 2018).

Phylogeography and demographic changes of Pseudonereis sp. East Asia

Pseudonereis sp. East Asia in the West Pacific off the Coast of China and Korea were supposed to be acknowledged in a single species. Based on the TCS network, the dominant haplotype composed of many sequence samples were derived from YS and ECS-TS. A high proportion of the number of private haplotypes were arisen by a few mutations. This implicates a lack of significant phylogeographic structuring in Pseudonereis sp. East Asia across the ECS off the Coast of Zhejiang and other geographically proximate areas in the West Pacific. One reasonable interpretation is that larval transportation and oceanographic connection via the longitudinal coastal currents along ECS-TS and YS (Avise et al. 1987; Grant & Bowen 1998), offset genetic differentiation driven by distinct ocean conditions or simply random effect of genetic drift across the species distributional ranges in the West Pacific.

The results of neutral test, mismatch distribution and Bayesian Skyline jointly suggested that the local and whole population of Pseudonereis sp. East Asia had a demography history of relatively steady population size followed some period of population growth in the recent history.

The reproductive mode in Pseudonereis sp. East Asia is probably via external fertilization, that typically results in longer dispersal distance than internal fertilization (Jackson 1986). It is likely that genetic exchange in Pseudonereis sp. East Asia along ECS-TS and YS are driven by the Taiwan Warm Current (south to north direction), ECS Coastal Current, YS Warm Current (south to north direction) and YS Cold Current (north to south direction; Feng et al. 1999). In addition, the Kuroshio Current that is the world's second largest warm current has a strong impact on the ocean circulation int the West Pacific. Reportedly, P. variegata was also found in as far north in the area of estuarine low-salinity of Japan (Sato 2017), yet no relevant molecular information is available for taxonomic and phylogeographic comparison. Supposing that Pseudonereis sp. East Asia occurs in the West Pacific off the Coast of Japan, these currents would play key roles in keeping them in the same species, including YS off the Coast of Korean Peninsula and Shandong and ECS-TS off the Coast of Zhejiang and Fujian.

Future research perspectives

Thus far, 1,000 species in 60 families in Polychaeta have been named in the coastal waters of East Asia (Wu et al. 1997). During the study, we found that a large proportion of polychaeteous annelids discovered in ECS areas are yet to characterize or confirmed at the molecular level. The same condition may occur in adjacent sea areas such as YS and the South China Sea. Further research endeavors on Polychaeta and other marine benthos are of great need not only using morphology, but also using molecular tools. Meanwhile, the nucleotide and barcode of life databases of polychaetes require significant improvement with more research investments that will help solve many taxonomic problems in marine biodiversity and environmental studies.

Our study provides basic scientific information for understanding the identity and genetic diversity of Pseudonereis sp. East Asia in the East China Sea off the Coast of Zhejiang, China and neighboring areas. Despite that, further examinations of Pseudonereis sp. East Asia are needed, in particular to examine morphological diagnostic characters and redescribe Pseudonereis sp. East Asia in comparison with the type specimen of P. variegata in Chile. In-depth collection of Pseudonereis sp. East Asia samples and information of their habitats will help cast light on the species’ population structure, phylogeography and adaptation in the distributional range of West Pacific on a greater seascape scale. Additional sequences of genes, e.g. mitochondrial ribosomal genes and/or nuclear genes derived from genomic data of Pseudonereis and other species in Nereididae will be beneficial (Aguado et al. 2007; Satheeshkumar & Jagadeesan 2010; Li et al. 2016; Paiva et al. 2019) to reveal ecological and genetic mechanisms of speciation.

Acknowledgement

We are grateful to Prof Houcai Cai, Wandong Chen, Li Lin, Erwei Wu, Xiaopin Ni and Shangwei Xie (Nanji Archipelago National Marine Nature Reserve Administration) for their assistance in sampling, and Rui Yao, Hui Zhou, Anqi Yan, Feiang Xie, Junlai Mao and Bowen Wu (Zhejiang Ocean University) for their assistance in sampling and data collection.

Funding

This study was supported by the Research Program of Nanji Archipelago National Marine Nature Reserve, China (NJKJ-2017-014) and Special Program for Basic Research of the Ministry of Science and Technology of China (2014FY110500).

Authors contributions

All authors contributed to the study conception and design. Y. C. and Z. M. K. conducted material preparation and data collection. Z. L. and Z. M. K. conducted data analyses. Z L prepared the first draft of the manuscript. Y. C. supervised and acquired funding for the project. Y. C. critically revised the manuscript. All the authors read and agreed to the final version of the manuscript.

Data Availability Statement

COI DNA sequences: GenBank accessions OR413633-OR413691.

Conflicts of Interest

The authors declare that they have no conflict of interest to declare.

Ethical Approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed by the authors.

Abdelnaby A F (2020) On some Nereididae (Polychaeta) with new records for the Egyptian waters. Egypt J Aquat Biology Fisheries 24:47–68
Abdel-Mawgood AL (2012) DNA based techniques for studying genetic diversity. InTech Rijeka, Croatia
Aguado MT, Nygren A, Siddall ME (2007) Phylogeny of Syllidae (Polychaeta) based on combined molecular analysis of nuclear and mitochondrial genes. Cladistics 23:552–564
Aljanabi SM, Martinez I (1997) Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques. Nucleic Acids Res 25:4692–4693
Avise JC, Arnold J, Ball RM, Bermingham E, Lamb T, Neigel JE, Reeb CA, Saunders NC (1987) Intraspecific phylogeography: the mitochondrial DNA bridge between population genetics and systematics. Annu Rev Ecol Syst 18:489–522
Bakken T (2007) Revision of Pseudonereis (Polychaeta, Nereididae). Zool J Linn Soc 150:145–176
Barnes RSK, Calow PP, Olive PJ, Golding DW, Spicer JI (2009) The invertebrates: a synthesis. John Wiley & Sons
Barroso R, Klautau M, Solé-Cava AM, Paiva PC (2010) Eurythoe complanata (Polychaeta: Amphinomidae), the ‘cosmopolitan’fireworm, consists of at least three cryptic species. Mar Biol 157:69–80
Beesley PL, Ross GJ, Glasby CJ (2000) Polychaetes & allies: the southern synthesis. CSIRO publishing
Bouckaert R, Vaughan TG, Barido-Sottani J, Duchêne S, Fourment M, Gavryushkina A, Heled J, Jones G, Kühnert D, De Maio N (2019) BEAST 2.5: An advanced software platform for Bayesian evolutionary analysis. PLoS Comput Biol, 15, e1006650
Brett CD (2006) Testing the effectiveness of the mt DNA Cytochrome c oxidase subunit 1 (COI) gene locus for identifying species of Polychaete worm (Polychaeta: Annelida) in New Zealand. The University of Waikato
Clement M, Snell Q, Walker P, Posada D, Crandall K (2002) TCS: estimating gene genealogies. Parallel and Distributed Processing Symposium. In: International Proceedings, p. 10.1109
Conde-Vela VM (2018) New species of Pseudonereis Kinberg, 1865 (Polychaeta: Nereididae) from the Atlantic Ocean, and a review of paragnath morphology and methodology. Zootaxa 4471:245–278
Daniels SR, Stewart BA, Cook PA (2002) Congruent patterns of genetic variation in a burrowing freshwater crab revealed by allozymes and mt DNA sequence analysis. Hydrobiologia 468:171–179
Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: more models, new heuristics and parallel computing. Nat Methods 9:772–772
Excoffier L, Laval G, Schneider S (2005) Arlequin (version 3.0): an integrated software package for population genetics data analysis. Evolutionary Bioinf 1:117693430500100003
Fauchald K (1977) The polychaete worms. Definitions and keys to the orders, families and genera. Natural History Museum of Los Angeles County, Science Series
Feng S, Li F, Li S (1999) An introduction to marine science. China Higher Education Press, Beijing, China
Fu Y-X (1997) Statistical tests of neutrality of mutations against population growth, hitchhiking and background selection. Genetics 147:915–925
Ge M, Xu Q, Fan S, Wang Z, Zhang X (2018) Taxonomy at order and family levels of the benthic groups of Polychaeta in the coastal waters of China. Biodivers Sci 26:998–1003
George JD, Hartmann-Schröder G (1985) Polychaetes: British Amphinomida, Spintherida and+ & Eunicida: Keys and Notes for the Identification of the Species. Brill Archive
Gibbs P (1969) Aspects of polychaete ecology with particular reference to commensalism. Philosophical Trans Royal Soc Lond Ser B Biol Sci 255:443–458
Gibbs P (1972) Polychaete annelids from the Cook Islands. J Zool 168:199–220
Giere O (2008) Meiobenthology: the microscopic motile fauna of aquatic sediments. Springer Science & Business Media
Grant W, Bowen BW (1998) Shallow population histories in deep evolutionary lineages of marine fishes: insights from sardines and anchovies and lessons for conservation. J Hered 89:415–426
Hall T (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. In: Nucleic Acids Symp Ser, pp. 95–98
Hebert PD, Ratnasingham S, De Waard JR (2003) Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proceedings of the Royal Society of London Series B: Biological Sciences, 270, S96-S99
Heled J, Drummond AJ (2008) Bayesian inference of population size history from multiple loci. BMC Evol Biol 8:1–15
Hutchings P, Kupriyanova E (2018) Cosmopolitan polychaetes–fact or fiction? Personal and historical perspectives. Invertebrate Syst 32:1–9
Hutchings P, Murray A (1984) Taxonomy of polychaetes from the Hawkesbury River and the southern estuaries of New South Wales. Australian Museum, Australia
Jackson J (1986) Modes of dispersal of clonal benthic invertebrates: consequences for species' distributions and genetic structure of local populations. Bull Mar Sci 39:588–606
Kara J, Macdonald AH, Simon CA (2018) Integrative taxonomic methods reveal an incorrect synonymisation of the South African Pseudonereis podocirra (Schmarda) as the widespread Pseudonereis variegata (Grube) from Chile. Invertebrate Syst 32:1282–1297
Kara J, Santos CS, Macdonald AH, Simon CA (2020) Resolving the taxonomic identities and genetic structure of two cryptic Platynereis Kinberg species from South Africa. Invertebrate Syst 34:618–636
Knowlton N (1993) Sibling species in the sea. Annu Rev Ecol Syst 24:189–216
Knowlton N (2000) Molecular genetic analyses of species boundaries in the sea. Marine genetics. Springer. p.^pp. 73–90
Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35:1547
Kvist S (2016) Does a global DNA barcoding gap exist in Annelida? Mitochondrial DNA Part A 27:2241–2252
Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R (2007) Clustal W and Clustal X version 2.0. bioinformatics, 23, 2947–2948
Leigh JW, Bryant D (2015) popart: full-feature software for haplotype network construction. Methods Ecol Evol 6:1110–1116
Li S, Chen Y, Zhang M, Bao X, Li Y, Teng W, Liu Z, Fu C, Wang Q, Liu W (2016) Complete mitochondrial genome of the marine polychaete, Marphysa sanguinea (Polychaeta, Eunicida). Mitochondrial DNA Part A 27:42–43
LI Y, Song-Hui L, JIANG T-J, LI H, XIAO Y-P, YOU S-P (2009) DYNAMICS OF PROROCENTRUM POPULATION AND ITS RELATION WITH ENVIRONMENTAL FACTORS IN NANJI ISLANDS SEA AREA. Acta Hydrobiol Sin, 02
Librado P, Rozas J (2009) DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25:1451–1452
Liu P, Yu Y, Liu C (1991) Studies on the situation of pollution and countermeasures of control of the oceanic environment in Zhoushan fishing ground—the largest fishing ground in China. Mar Pollut Bull 23:281–288
Lobo J, Teixeira MA, Borges LM, Ferreira MS, Hollatz C, Gomes PT, Sousa R, Ravara A, Costa MH, Costa FO (2016) Starting a DNA barcode reference library for shallow water polychaetes from the southern European Atlantic coast. Mol Ecol Resour 16:298–313
Loeza-Quintana T, Carr CM, Khan T, Bhatt YA, Lyon SP, Hebert PD, Adamowicz SJ (2019) Recalibrating the molecular clock for Arctic marine invertebrates based on DNA barcodes. Genome 62:200–216
Mantel N (1967) The detection of disease clustering and a generalized regression approach. Cancer Res 27:209–220
Neira C, Höpner T (1994) The role of Heteromastus filiformis (Capitellidae, Polychaeta) in organic carbon cycling. Ophelia 39:55–73
Nygren A (2014) Cryptic polychaete diversity: a review. Zoolog Scr 43:172–183
Paiva PC, Mutaquilha BF, Coutinho MCL, Santos CS (2019) Comparative phylogeography of two coastal species of Perinereis Kinberg, 1865 (Annelida, Polychaeta) in the South Atlantic. Marine Biodivers 49:1537–1551
Peakall R, Smouse PE (2006) GENALEX 6: genetic analysis in Excel. Population genetic software for teaching and research. Mol Ecol Notes 6:288–295
Pettibone MH (1982) Annelida. Synopsis and Classification of Living Organisms, Volume 2
Pettibone MH (1986) Additions to the family Eulepethidae Chamberlin (Polychaeta: Aphroditacea). Smithsonian Contributions to Zoology
Puillandre N, Lambert A, Brouillet S, Achaz G (2012) ABGD, Automatic Barcode Gap Discovery for primary species delimitation. Mol Ecol 21:1864–1877
Rambaut A, Drummond AJ, Xie D, Baele G, Suchard MA (2018) Posterior summarization in Bayesian phylogenetics using Tracer 1.7. Systematic biology, 67, 901–904
Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572–1574
Rozbaczylo N, Bolados J (1980) Néridos de Iquique. Nereidae, Chile (Polychaeta
Sampertegui S, Rozbaczylo Narváez N (2013) Morphological and molecular characterization of Perinereis gualpensis (Polychaeta: Nereididae) and its phylogenetic relationships with other species of the genus off the Chilean coast, Southeast Pacific
Satheeshkumar P, Jagadeesan L (2010) Phylogenetic position and genetic diversity of Neridae—polychaeta based on molecular data from 16S r RNA sequences. Middle East J Sci Res 6:550–555
Sato M (2017) Nereididae (Annelida) in Japan, with special reference to life-history differentiation among estuarine species. Species diversity of animals in Japan. Springer p ^pp 477–512
Scheltema RS (1968) Dispersal of larvae by equatorial ocean currents and its importance to the zoogeography of shoal-water tropical species. Nature 217:1159–1162
Scheltema RS (1971) Larval dispersal as a means of genetic exchange between geographically separated populations of shallow-water benthic marine gastropods. Biol Bull 140:284–322
Scheltema RS (1988) Initial evidence for the transport of teleplanic larvae of benthic invertebrates across the East Pacific Barrier. Biol Bull 174:145–152
Schmidt H, Westheide W (2000) Are the meiofaunal polychaetes Hesionides arenaria and Stygocapitella subterranea true cosmopolitan species?—results of RAPD-PCR investigations. Zoolog Scr 29:17–27
Silva CF, Seixas VC, Barroso R, Di Domenico M, Amaral AC, Paiva PC (2017) Demystifying the Capitella capitata complex (Annelida, Capitellidae) diversity by morphological and molecular data along the Brazilian coast. PLoS ONE, 12, e0177760
Sun R, Yang D (2004) Fauna sinica, invertebrate. Annelida, Polychaeta (II), Nereidida, 33, 30–41
Tajima F (1989) Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123:585–595
Tang Y, Liao Y, Shou L, Zeng J, Gao A, Chen Q, Sun Q (2014) Influence of coralline algae on biodiversity of macrobenthic community in intertidal zone of Nanji Islands. Biodivers Sci 22:640
Tornabene L, Baldwin C, Weigt LA, Pezold F (2010) Exploring the diversity of western Atlantic Bathygobius (Teleostei: Gobiidae) with cytochrome c oxidase-I, with descriptions of two new species. aqua 16:141–170
Tosuji H, Sato M (2010) Genetic evidence for parapatric differentiation of two forms of the brackish-water nereidid polychaete Hediste atoka. Plankton and Benthos Research 5:242–249
Van Herwerden L (1989) Collection of mussel worms Pseudonereis variegata for bait—a legislative anachronism. S Afr J Mar Sci 8:363–366
Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol 3:294–299
Wu B, Wu Q, Qiu J, Lu H (1997) Fauna Sinica Invertebrata (Vol. 9) Polychaeta: Phyllodocimorpha. Science Press, Beijing
Wu P-l, Sun J-p, Yang T-c (1985) Nereidae (Polychaetous annelids) of the Chinese coast. Springer-Verlag
Yang D, Sun R (1988) Polychaetous Annelids of the Chinese Coast (ä¸å›½è¿‘æµ·å¤šæ¯›çŽ¯èŠ‚åŠ¨ç‰©). China Agriculture Press, Beijing.(in Chinese
Zhang J, Kapli P, Pavlidis P, Stamatakis A (2013) A general species delimitation method with applications to phylogenetic placements. Bioinformatics 29:2869–2876
Zhang Z, Schwartz S, Wagner L, Miller W (2000) A greedy algorithm for aligning DNA sequences. J Comput Biol 7:203–214

Download PDF

Version 1

posted

You are reading this latest preprint version

Taxonomic status and phylogeographical structure in Pseudonereis sp. East Asia in the East China Sea off the Coast of Zhejiang, China

Status:

Version 1

Abstract

Figures

Introduction

Material and methods

Sample collection and morphological identification

DNA extraction, PCR, and DNA sequencing

Data analysis

Results

Morphology

Sequence polymorphisms

Phylogenetic and delimitation analysis

Haplotype network and demography history

Discussion

Future research perspectives

Declarations

References

Status:

Version 1