¹ School of Marine Sciences, Ningbo University, Ningbo 315211, P. R. China² Key Laboratory of Marine Ecosystem Dynamics, Second Institute of Oceanography, Ministry of Natural Resources, Hangzhou 310012, P. R. China³ Observation and Research Station of Yangtze River Delta Marine Ecosystems, Ministry of Natural Resources, Zhoushan 316022, P. R. China* Correspondence: lichenghua@nbu.edu.cnAbstract: Sea-level fluctuations in the marginal seas of the northwestern Pacific Ocean during the Pleistonene have profoundly influenced the genetic structure of marine organisms. Previous phylogeographic studies have mainly focused on fish and molluscs; other taxonomic groups have been poorly studied, which restricts a comprehensive understanding of the geogenetic patterns of marine species in this area. To precisely understand how environmental factors and historical events shape the population structure of the sea penCavernularia obesa in the East China Sea, we determined partial nucleotide sequences of the mitochondrial cytochrome oxidase subunit I (COI) gene in 71 individuals from five sites. Results of population Genetic and demographic analyses revealed a low genetic diversity within each population. Moreover, the historical population size change showed that the populations experienced bottlenecks during the Pleistonene. The sea level of the East China Sea decreased by >100 m when the glacial advanced during the Pleistonene, which caused the shrinking of habitats for marine species and eventually resulted in low genetic diversity within the populations in this area.Keywords: phylogeography; marginal sea; Cavernularia obesa; sea level fluctuation; Changjiang diluted water 1. Introduction Phylogeography is the study of the geographic distribution of genetic lineages within a species and provides a means for inferring historical and contemporary processes that influence population distribution and abundance. The phylogeographic distribution of marine species is driven by historical events and modern environmental factors(Dong et al., 2012; Lima-Junior et al., 2021). Among these factors, oscillations of climate and oceanography have caused drastic changes in subtropical and temperate coastal environments(Avise, 2000). The marginal seas of the northwestern Pacific Ocean experienced sea-level fluctuations during late Quaternary glacial cycles(Wang, 1999). When the glacier advanced during the Pleistonene, the East China Sea (ECS) declined by approximately 130–150 m than present (Waelbroeck et al., 2002). The ECS shelf was completely exposed as the coastline migrated approximately 1200 km seaward (Wang, 1999). Consequently, the ECS was reduced to an elongated enclosed sea ( Okinawa Trough) with an area <1/3 of its present size (Ni et al., 2014). During interglacial periods, the ECS was inundated by rising sea levels. These repeated transgression–regression cycles, together with ocean currents and recent anthropogenic activities, have greatly impacted the phylogeographic patterns and population genetic differentiation in this area. Therefore, the ECS has been proposed as an ideal model for studying how sea level oscillations are caused by repeated glaciations, structured distribution ranges, spatial population genetic diversity, and marine phylogeography (Ni et al., 2014). The Changjiang River is the largest river in Asia and the third-largest river in the world. It enters the ECS with about 9×10¹¹ cm³ of freshwater discharge annually. This huge freshwater flow, named the Changjing diluted water (CDW), causes significant shifts in various ecological and environmental parameters in the ECS and acts as a barrier to the genetic connectivity of marine species. In other marine realms, some well-known biogeographic boundaries, such as the Basic Isthmus(York et al., 2008), Central American Isthmus(Coppard and Lessios, 2017), and Cape Hatteras(Mccartney et al., 2013), have proven to be intraspecific barriers restricting the gene flow of some wide-ranging taxa. Recently, several studies have tested the phylogeographic barrier effect of CDW in addition to biogeographic boundary[1,10-12]. However, the results of these studies are inconsistent, and controversy has arisen as different genetic patterns have emerged. Previous phylogeographic studies on the ECS have mainly focused on commercially exploited fish and molluscs[12,13]. Although the genetic structure of commercial species may be affected by invasion genomics(Jaspers et al., 2021; Rius and Turon, 2020), species from other diverse taxonomic groups are good candidates for such comparative studies(Ni et al., 2017). The sea pen C. obesa is a widely distributed colonial cnidarian found in the northeastern Pacific Ocean (López-González et al., 2000). Sea pens undergo a two-phase life cycle of planktonic larvae and sessile adult forms. Mature adults of C. obesa half-buried in the sediments of the seabed and barely move, and their dispersion mainly relies on planktonic larvae. The commercial value of C. obesa is low; therefore, the genetic structure of the ECS community was unaffected by commercial aquaculture activities. These characteristics make C. obesa an ideal model species for phylogeographic studies of the ECS. In this study, we collected 71 individuals of C. obesa from five sites in the ECS and used phylogenetic analyses based on mitochondrial cytochrome oxidase subunit I (COI) to analyze the range-wide phylogeographic structure and determine the potential drivers of spatial genetic variation in C. obesa. 2. Materials and Methods 2.1. Sample collection A total of 71 individuals of C. obesa were collected from five sites using Agassiz trawl in the ESC from 2016 to 2019. The samples were frozen at -20 °C. As C. obesa is not an endangered or protected species, and collection was only carried out from public access areas, no specific permits were required to collect this species from these locations. The locations of the sampling sites are shown in Figure 1 and Table 1.Figure 1. Map of Northwest Pacific showing sampling sites and haplotypes of C. obesa; YSCC, Yellow Sea Coastal Current; CDW, Changjiang Diluted Water; and ECSCC, East China Sea Coastal CurrentTable 1. Sampling sites and diversity indices for the five populations of C. obesa