Three new species of Heteromastus (Annelida, Capitellidae) from Korean waters, with genetic evidence based on two gene markers

Abstract Three undescribed species of Heteromastus Eisig, 1887 were collected from intertidal to sublittoral habitats in western and southern waters of Korea. Heteromastus namhaensis sp. nov. is distinguishable from other congeners by the presence of hemispheric notopodial lobes in the posterior abdomen. Heteromastus gusipoensis sp. nov. closely resembles H. tohbaiensis Yabe & Mawatari, 1998 in the absence of posteriorly extended abdominal notopodial lobes, but differs in the absence of eyespots on the prostomium and distinct node on the shaft of thoracic hooks in H. gusipoensis. Heteromastus koreanus sp. nov. is similar to H. filiformis sensu Hutchings & Rainer, 1982 in the shape of abdominal notopodia, but clearly differs in dentition of the abdominal hooks and methylene green staining pattern (MGSP). DNA sequences (mtCOI and histone H3) of these new Korean species were compared with all sequences of Heteromastus species available in the public database. Molecular results showed distinct genetic differences among these three new Korean species at species level. Comparison of mtCOI gene revealed significant genetic difference between H. filiformis and these Korean species. A comprehensive comparison between three Heteromastus species of present study and their closely related congeners is conducted based on morphological and genetic results.


Introduction
The genus Heteromastus Eisig, 1887, which belongs to the family Capitellidae Grube, 1862, is commonly found from intertidal areas to shallow subtidal depths in a variety of sediment types, including fine and silty sand and mud (Blake 2000;Dean 2001). Feeding activity of Heteromastus plays an important role in the supply of overlying oxygenated water into anoxic muds below the redox potential discontinuity (Cadée 1979). Heteromastus is known as a biological indicator and opportunistic species in marine hypoxia condition (Cadée 1979). The genus Heteromastus was first designated by Eisig (1887) based on the description of H. filiformis Claparède, 1864 (as Capitella filiformis) from southern France. According to his diagnosis, Heteromastus is distinguished from other genera in the family by having 11 thoracic chaetigers, of which the first five only have capillaries. Green (2002) improved this generic definition by including the differences in the thoracic (long-shafted) and abdominal (short-shafted) hooks. However, the lack of good generic characteristics has led to taxonomic confusion in this genus (Blake 2000;Green 2002). For instance, although Heteromastus currently contains seven valid species (Read and Fauchald 2018), the chaetal arrangement of H. giganteus Zachs, 1933 does not match to the original generic definition (Zachs 1933). Among the recognized generic characteristics, the number of thoracic segments can be miscounted due to ambiguous boundaries among peristomium, thorax, and abdomen (Blake 2000). In addition, the thoracic chaetal arrangement varies depending on the degree of development (Fredette 1982).
Heteromastus filiformis (Claparède, 1864), the generic type species, is well known as a cosmopolitan species found in various types of the habitats and has been referred to in many ecological studies (Hutchings and Rainer 1982). Species-specific characters of this representative species have been controversial due to incomplete original description and the absence of the original type specimens from southern France, although Hutchings and Rainer (1982) have later designated the neotype from Egypt (Green 2002). In addition, the dental structure of abdominal hooks and the shape of posterior parapodial lobes of H. filiformis have been described differently in published records including the neotype (Blake 2000;Green 2002). In Korean waters, Choi and Yoon (2016) have reported that H. filiformis was the only species belonging to genus Heteromastus occurring in this region based on morphological features. They have suggested that Korean specimens have some minor differences with former records of H. filiformis in the morphology of abdominal hooks and methylene green staining pattern, although these characters have been used for identification of recorded species in family Capitellidae (Blake 2000;Green 2002;Jeong et al. 2017b). Recently, a combination of morphological and molecular analysis has been conducted to distinguish very close polychaete species and geographical populations (e.g. Glasby et al. 2013;Jeong et al. 2017bJeong et al. , 2018. The aim of the present study is to verify the taxonomic status of undescribed Heteromastus species inhabiting Korean waters based on morphological and molecular analysis using two different partial genes (mtCOI and histone H3) in comparison with their closest species in the genus.

Morphological analysis
Samples were collected from eight stations of Korean sublittoral areas using a 0.05 m 2 Van Veen grab (Fig. 1). Sediment samples were elutriated over a 0.5 mm sieve in a 30 L seawater container and organisms were transferred to a 1 L collecting jar containing 7% MgCl 2 solution for anesthesia. Relaxed samples were fixed in a buffered solution of 10% formalin within one hour and then finally preserved in 95% ethanol. In the laboratory, Heteromastus specimens were sorted under a Zoom Stereomicroscope (SMZ745T, Nikon). Line drawings were performed using a differential interference contrast microscope (Eclipse Ci-L, Nikon) and a digital pen display (Cintiq 22HD, Wacom). Methyl green staining patterns (MGSP) and scanning electron microscopy (SEM) analyses were performed as delineated by Jeong et al. (2017b). The examined type materials were deposited in the collection of Marine Biodiversity Institute of Korea (MABIK) in Seocheon, Korea (Table 1).

Molecular analysis
Genomic DNA was extracted from ethanol-preserved specimens. Specimens used for molecular analysis were partially dissected (ca 2 segments) in the middle part of the abdomen. To extract genomic DNA, 1.5 mL centrifuge tubes each containing 45 μL of 10% Chelex suspension (Bio-Rad Laboratories Inc.), 5 μL of Proteinase K (10 mg/ml, iNtRON Biotechnology, Inc.), and dissected tissues (ca 2 segments) were incubated at 56 ℃ for 3-12 hours. Extracted genomic DNA was used as a template to amplify the target region. Polymerase chain reaction (PCR) was performed on a MasterCycler PCR thermal cycler (Eppendorf Co.). The primer pair for mtCOI was LCO1490 and HCO2198 (Folmer et al. 1994). For histone H3, it was H3F and H3R (Colgan et al. 1998). PCR mixtures contained 17 μL of deionized water, 1 μL of each primer (10 μM), 1 μL of DNA template and PCR premix (20 μL, BiONEER Co.  Table 1 summarizes information for all sequences used in the analyses. These aligned sequences were used as data sets to generate genetic distance using Kimura's twoparameter (K2P) model (Kimura 1980). Based on K2P distances, intraspecific genetic differences within the Korean specimens and the interspecific genetic differences among the closest taxa were calculated.
Abdominal parapodial lobes located in posterior end of each segment, well separated from each other, and gradually developed posteriorly ( Fig. 2A-D). Abdominal notopodia separated, mid-dorsal on anterior few segments, becoming dorsolateral in following abdominal region, with six to eight hooded hooks per fascicle, having dorsoposteriorly protruded and hemispheric lobes from chaetiger 90 to end of body (Figs 2A-D, 5B). Abdominal neuropodia well separated, with 10-12 hooded hooks per fascicle, having slightly protruded lobes in posterior abdomen; neuropodial lobes less developed than notopodial lobes (Figs 2C, D, 5B).
Hooded hooks with main fang extending slightly beyond hoods. Abdominal hooks with distinct node on shaft and four rows of small teeth above main fang; three teeth in basal row, three in second and third row, and four to six in superior row (Figs 2E, G, 5A). Pygidium with digitate anal cirrus (Figs 2D, 5B).
Etymology. The species is named for its wide distribution in Namhae (=Korean name of southern sea of Korea). Remarks. Heteromastus namhaensis resembles H. filiformis sensu Hutchings & Rainer, 1982 in the absence of distinct eyespots on prostomium, three teeth in basal row above the main fang of abdominal hooks, and the presence of posteriorly extended abdominal notopodial lobes (Table 2). However, they differ in the shape of notopodial lobes in posterior abdomen (hemispheric protrusion in H. namhaensis vs broadly-based and rounded lamellae in H. filiformis sensu Hutchings & Rainer, 1982), the different dental structure of abdominal hooks (Table 2). Heteromastus namhaensis is also easily distinguished from Korean former record of H. filiformis (Choi and Yoon 2016) by the presence of hemispheric abdominal parapodial lobes and the absence of eyespots in H. namhaensis. In particular, the hemispheric notopodial lobe of H. namhaensis is a unique feature in the genus. Diagnosis. Abdominal hooks with four rows of teeth; three teeth in basal row, three in second row, four in third row, and two in superior row. Genital pores present in intersegmental furrows of between chaetigers 5-6, 6-7, 7-8, 8-9, 9-10, and 10-11. Posteriorly extended parapodial lobes absent on abdominal segments.
Hooded hooks with main fang extending slightly beyond hoods. Abdominal hooks with distinct node on shaft and four rows of small teeth above main fang; three teeth in basal row, three in second row, four in third row, and two in superior row (Figs 3E, G, 5C). Pygidium with digitate anal cirrus (Figs 3D, 5D).
Etymology. The new species is named for its limited distribution in Gusipo, Korea. Distribution. Intertidal area (0-1 m) near Gusipo, Korea. Ecology. Heteromastus gusipoensis was sampled in May of 2015 (9 ind./m 2 ) and November of 2017 (71 ind./m 2 ). Most well-developed individuals (having over 120 segments) were obtained in November. Surface sediment of the collecting station was mainly composed of fine sand and silt. Unidentified nereidid polychaetes co-occurred in the same location. The salinity of the sampling location was about 32.
Remarks. Heteromastus gusipoensis closely resembles H. tohbaiensis Yabe & Mawatari 1998 in the chaetal arrangement and the absence of developed parapodial lobes in posterior abdomen (Table 2). However, they differ in the presence of eyespots on prostomium and distinct node on the shaft of thoracic hooks in H. tohbaiensis (Table 2;

Diagnosis.
Abdominal hooks with three rows of teeth; two teeth in basal row, three in second row, and four in superior row. Genital pores present in intersegmental furrows between chaetigers 7-8, 8-9, 9-10, and 10-11. Posteriorly extended and rounded thin parapodial lobes present on posterior abdominal segments.
Transition between thorax and abdomen distinguished by changes in shape of chaetae and segment (Fig. 4A); anterior abdominal segments multi-annulated, gradually longer posteriorly, with short-shafted hooded hooks placed posteriorly in segment; posterior thoracic chaetigers bi-or tri-anullated, with long-shafted hooded hooks in central part of segment; last thoracic chaetiger smaller than first abdominal chaetiger (Fig. 4A). Abdominal parapodial lobes located in posterior end of each segment, well separated from each other, and gradually developed posteriorly (Fig. 4C, D). Abdominal notopodia separated, middorsal on anterior few segments, becoming dorsolateral in following abdominal region, with 5 or 6 short-shafted hooded hooks per fascicle, having posteriorly extended and rounded thin lobes from chaetiger 70-80 to end of body; expanded notopodial lobes overlap dorso-anterior part of further segment (Figs 4C, D, 5F). Abdominal neuropodia well separated, with 10-12 short-shafted hooded hooks per fascicle, having slightly protruded lobes in posterior abdomen; neuropodial lobes less developed than notopodial lobes (Figs 4C, D, 5F).
Hooded hooks with main fang extending slightly beyond hoods. Abdominal hooks with distinct node on shaft and three rows of small teeth above main fang; two teeth in basal row, three in second row, and four in superior row (Figs 4E,G,5E). Pygidium with digitate anal cirrus (Fig. 4D).
Etymology. The new species is named for its wide distribution in coastal waters of Korea.
Distribution. Intertidal areas (0-1 m) near Korea (Fig. 1). Ecology. Heteromastus koreanus was mainly sampled from Gwangyang in April of 2014 (35 ind./m 2 ) and November of 2017 (470 ind./m 2 ). Most well-developed individuals (having over 110 segments) were obtained from Muan and Gwangyang in November and coelomic eggs were 54-71 μm in diameter. Surface sediment of the collecting station was mainly composed of fine sand and silt. Unidentified cirratullid and nereidid polychaetes co-occurred in Gwangyang, Korea. The salinity range among sampling locations was about 15-33. Gwangyang is the only estuarine habitat. Other locations are situated in marine mud flats.
Remarks. Heteromastus koreanus closely resembles former records of H. filiformis reported by Hutchings and Rainer (1982) and Choi and Yoon (2016) in the chaetal arrangement, the presence of posteriorly extended notopodial lobes in posterior abdomen, and the absence of the spine-like uncini and the distinct branchial structure (i.e. filamentous or digitiform) in posterior abdomen (Warren 1994;Blake 2000; Table  2). However, they differ in the dentition of abdominal short-shafted hooks (2/3/4 in H. koreanus vs 3-4/4-5/4-6 in H. filiformis sensu Hutchings & Rainer, 1982 vs three or four teeth in three rows in H. filiformis sensu Choi & Yoon, 2016), and the speciesspecific MGSP (Table 2). Additionally, H. filiformis occurs in the marine intertidal areas of Atlantic, Mediterranean, and America (Blake 2000) whereas H. koreanus of present study is collected mainly from the estuarine environment (salinity of 15-23) of Korea (Table 2). Heteromastus koreanus is also similar to H. tohbaiensis in the chaetal arrangement and presence of eyespots. However, they clearly differ in absence of distinct node on shaft of thoracic hooks and presence of expanded abdominal parapodial lobes in H. koreanus (Yabe 1998).

Molecular comparisons.
To verify the genetic divergence between examined specimens, partial sequences of mitochondrial (mtCOI) and nuclear (histone H3) genes were used. Intraspecific differences for mtCOI (MK032276-MK032284) and histone H3 (MK032285-MK032293) genes of each Korean species were very low (0-0.4%, Table 3). Based on mtCOI gene comparison, mean interspecific differences among these three new Korean species of the present study were distinct (16.0-18.9%, Table 3). All examined Korean Heteromastus species were well distinguished genetically from H. filiformis of China (13.3-19.6%, HZPLY183-12) and America (19.7-22.0%, MH235890). Based on histone H3 gene comparison, mean interspecific differences among the Korean Heteromastus species were 2.8-5.4% (Table 3). The known genetic difference for the mtCOI gene among capitellid species is 12.3-23.7% (Jeong et al. 2017b). In contrast, the published histone H3 gene difference between cryptic polychaetes is 2-9% (Glasby et al. 2013). Thus, genetic differences of these examined Heteromastus species (COI: 13.3-22.0%, H3: 2.8-5.4%) are significant at species level. Among all sequences of unidentified Heteromastus in Genbank database, sequences regarding two specimens from southern Japan (COI: LC208123-LC208124, H3: LC208100-LC208101) were genetically very close to H. koreanus of present study (COI gene difference: 2.1-3.3%, H3 gene difference: 0.9-1.3%). Among the described Heteromastus species from Japan, H. tohbaiensis resembles H. koreanus in the chaetal arrangement and presence of prostomial eyespots. However, they clearly differ in presence of distinct node on shaft of thoracic hooded hooks and absence of expanded abdominal parapodial lobes in H. tohbaiensis (Yabe 1998). Moreover, these two unidentified sequences (LC208123-LC208124) were originally reported from tidal mud flat and estuary near southern Japan, respectively (Tomioka et al. 2018). This distribution pattern is similar with those of H. koreanus (i.e. wide salinity range of 15-33) rather than H. tohbaiensis, which have been reported from lacustrine habitat of northern Japan. Despite the lack of morphological information regarding these Japanese specimens, the high similarity in genetic feature and inhabiting environment confirms the additional occurrence of H. koreanus in southern Japan.