Review of the genus Loimia Malmgren, 1866 (Annelida, Terebellidae) from China seas with recognition of two new species based on integrative taxonomy

Specimens of the genus Loimia (Annelida, Terebellidae) deposited in the Marine Biological Museum of the Chinese Academy of Sciences (MBMCAS) together with materials newly collected from China seas were examined. Based on morphological comparisons and molecular analysis, some specimens collected from the coasts of Shandong province and Guangxi province were confirmed as two new Loimia species respectively (Loimia borealis sp. n. and Loimia macrobranchia sp. n.). Morphologically, L. borealis sp. n. is distinguished from previously known species of this genus by having seven equal sized ventral shields, with length five times the width; this species was retrieved as sister to the clades of Loimia arborea Moore, 1903 and Loimia bandera Hutchings, 1990 in the phylogenetic tree, which was reconstructed based on mitochondrial COI gene. Loimia macrobranchia sp. n. differs from congeners by the large size of its first pair of branchiae with a thick main stem and about 18 dendritic branches arranged in two levels. A key to identifying Loimia species found in Chinese seas is given.


Sample collection and morphological examination
Specimens of Loimia were collected from coastal areas of Chinese seas (Table 1). The collected specimens were anesthetized with 7% magnesium chloride (MgCl2) and photographed using a Canon EOS 600D camera. Thereafter they were preserved in 75% ethanol. All specimens were deposited in the MBMCAS. Specimens were observed, measured, and photographed with a Nikon SMZ25 stereomicroscope. The detailed morphology of the lobes, notochaetae, and uncini was observed and documented with a Loimia bandera Hutchings, 1990 China, Taiwan

Molecular data and analysis
DNA was extracted from 17 individuals collected from various coastal areas of Chinese seas that represented five morphologically distinct species of the genus Loimia (Table 1). Total DNA was extracted with the DNeasy Ò Blood and Tissue Kit (Qiagen, Hilden, Germany) and stored at −20 C. Partial fragments (approximately 700 bp) of the COI gene were amplified by the polymerase chain reaction (PCR) using primers LCO1490/ HCO2198, LCO1490/CO1-E, polyLCO/polyHCO, and polyLCO/polyshortCOIR. The primer information is given in Table 2. Amplifications were carried out in a reaction mixture containing 2 µl of template DNA, 12.5 µl of Premix Taq TM (Takara, Otsu, Shiga, Japan), 0.5 µl of each primer (stock concentration, 10 mM), and sterile distilled H 2 O to a total volume of 25 µl with cycling conditions as follows: initial denaturation at 94 C for 10 min, followed by 35 cycles of denaturation at 94 C for 30 s, annealing at 45 C for 40 s, and extension at 72 C for 30 s. A final extension at 72 C for 10 min was included. PCR-products generating distinct bands after electrophoresis on 1% agarose gels were sent to the Qingke Laboratory (Qingdao, China) for sequencing using the same set of primers that was used for PCR. Fragments with overlapping sequences (forward and reverse) were merged into consensus sequences using CONTIG EXPRESS (a component of Vector NTI Suite 6.0, Life Technologies, Carlsbad, CA, USA). The assembled sequences were checked by searching BLAST in GenBank to ensure that the DNA was not contaminated.
In addition to the sequences obtained by PCR, we downloaded all COI gene sequences of Loimia species from GenBank. COI gene sequences of two species from the family Terebellidae, namely Pista cristata Müller, 1776 andTerebella lapidaria Linnaeus, 1767 were also downloaded as the outgroups for phylogenetic analysis (Table 1).
Molecular data, including 31 sequences of the COI gene, were aligned using MUSCLE 3.8 (Edgar, 2004). Highly divergent and poorly aligned sites were omitted from the alignment according to Gblocks 0.91b (Castresana, 2000). The best-fitting nucleotide base substitution model (GTR+F+I+G4) for the alignment data was determined in IQTREE with ModelFinder (Kalyaanamoorthy et al., 2017), and a maximum likelihood tree was constructed using IQTREE (Nguyen et al., 2015) with 1,000 bootstrap reiterations  (Hoang et al., 2018). A Bayesian inference tree was constructed using MrBayes 3.2 (Huelsenbeck & Ronquist, 2001). Markov chains were run for 10,000,000 generations and sampled every 100 generations. The first 25% of trees were discarded as burn-in, and the remaining trees were used to construct the 50% majority-rule consensus tree and to estimate posterior probabilities. Genetic distances were calculated using the Kimura's two-parameter model in MEGA 7.0 (Kumar, Stecher & Tamura, 2016). Finally, all the sequences obtained in this study were submitted to GenBank (Table 1).

Zoobank registration
The electronic version of this article in portable document format will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank Life Science Identifiers (LSIDs) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix http://zoobank.org/. The LSID for this publication is: urn:lsid: zoobank.org:pub:06667411-789A-49C9-AC12-8DA2858341E9. The online version of this work is archived and available from the following digital repositories: Peer J, PubMed Central, and CLOCKSS.
Remarks. Loimia species are normally found in tropical areas, with only a few species inhabiting subtropical areas, and no species inhabiting Arctic areas (Lavesque et al., 2017).
The genus Loimia can be distinguished by the presence of pectinate uncini, a feature unique to Terebellidae (Nogueira, Hutchings & Carrerette, 2015). Other characteristics useful in the identification of species within this genus are: (1) the shape and position of the lobes on segments 1 and 3; (2) the number, arrangement, and shape of the mid-ventral glandular shields; and (3) the number and morphology of uncinial teeth from both anterior and posterior regions (Hutchings & Glasby, 1988, 1995Hutchings, 1997;Londoño-Mesa & Carrera-Parra, 2005;Londoño-Mesa, 2009;. Loimia borealis sp. n. Figs  Neuropodia starting from segment 5, as glandular ridges slightly raised from surface of thorax until segment on which notopodia terminate, thereafter as elongate pinnules (Figs. 2B, 2C, 2F and 3A). Uncini pectinate, with 5-6 teeth, higher than long, with oblique and concave base presenting slight protuberance; short triangular heel directed backwards for the attachment of the long, ligamental filament, with prow downwardly directed, aligned with line of teeth, connected to long filament (Figs. 4A-4D and 4G); under SEM, series of teeth with lateral fringe of minute teeth (Figs. 3G-3J). Uncini arranged in single row until segment 10, in double rows from segment 11-20, in back to back position; in single rows from segment 21 to the pygidium; abdominal uncini similar in shape but smaller in size than thoracic ones. Anus surrounded by about 7 rounded papillae. Tube with an inner lining of mucus covered by small stones and shell fragments. Variation. The number of segments in complete specimens varies from 95 to 104. The number of ventral shields varies from 7 to 9. Genital papillae absent in some specimens. In addition, other morphological characteristics used for comparison are provided in Table 3.
Remarks. Loimia borealis sp. n. is characterized by its compact rectangular mid-ventral shields, each with a length five times the width, which to our knowledge are not found in any other species in the genus Loimia. Loimia arborea and L. bandera also have slender ventral shields, but they differ from L. borealis sp. n. in the shape of their broad prominent prostomia, which almost surround the mouth (Figs. 5A, 5C and 5E), and their round glandular tubercles, which run in longitudinal ridges on anterior segments near notopodia (Figs. 5B, 5D and 5F) (Hutchings, 1990;Moore, 1903). Similar to L. borealis sp. n., Loimia ingens has large round lobes on segment 3 that originate dorso-laterally and align to the base of notopodia of segment 4 (Figs. 5H and 5J). However, L. borealis sp. n. has three pairs of equal-sized branchiae, which distinguish it from L. ingens (the first pair is the longest, and the third pair is the shortest) (Fig. 5I). These species are compared in Table 4.
Etymology. "Borealis" means northern. The species is named after its location on the northern coast of China.    Description of holotype. Specimen complete, with 125 segments, 358.9 mm long; thorax 15.7 mm long. Body yellowish brown in live material, with greenish branchiae and yellowish inclusions. Darker, reddish and yellowish mid-ventral shields terminating with blood red glandular region subdivided as transverse bands (Fig. 6A). Preserved body whitish, without distinct patterns of pigmentation (Figs. 6B-6E). Posterior thoracic segments longer than anterior segments; body swollen anteriorly, widest over mid posterior thorax, tapering gradually over abdominal segments (Fig. 6A). Notopodia beginning from segment 4 followed by 17 chaetigerous segments. Prostomium attached to dorsal surface of upper lip; basal part without eyespots; distal part almost straight, from which buccal tentacles originate. Prostomium broad, almost surrounding the mouth (Fig. 6D); upper lip with long free edge, projecting forward, shorter than wide, thrown into several vertical folds with the margins corrugated (Figs. 6C and 6D). Peristomium ventrally and laterally with a conspicuous free anterior border, ending above in a pair of narrow lobes originating dorsal-laterally and reaching around 1/3 of the upper lip length, mid-ventrally connected by membrane (Figs. 6C and 6D). Lower lip small, button like to rectangular, partly hidden by the membrane the first pair of lateral lobes (Fig. 6D). Segment 2 reduced, dorsally conspicuous, fuzed with segment 3, laterally forming a pair of small and triangular lobes (Fig. 6C). Lobes with bases wider than tips, dorsal margins 5-6 × 6 4-5 × 4-5 5-6 × 5-6 6×6 5-6 × 5-6 5-6 × 5-6 6×5-6 5 -6 × 5 5-6 × 5-6   aligned and fuzed to the notopodia of segment 4 (Figs. 6C and 6D). Segment 4 without lateral lobes. Three pairs of branchiae on segments 2-4 with yellowish inclusion (Figs. 6B and 6C). First pair of branchiae large, about 3 times as large as subsequent ones, with stout main stem and about 18 dendritic branches arranged in two levels on the main stem (Fig. 6E). The other two pairs of branchiae being subequal. Genital papillae small, at base of notopodia of the segments 6-8 (Fig. 6B). Ten ventral shields, starting from segment 2 to segment 12 (Fig. 6A); the first shield on segments 2-4 completely fuzed into single crenulated structure, being twice as long as following segments (Fig. 6D); anterior shields rectangular, all about the same size (Fig. 6D); behind segment 11, the glandular region subdivided as transverse bands, blood-red in live material (Fig. 6A). Bands on segment 12 and 13 trapezoid, width being twice as long as anterior shields; on segment 14 and 15 the largest, almost oval in outline; on segment 16 smaller than that on 15, oval in outline; subsequently bands becoming shorter and narrower, disappearing by segment 19 (Fig. 6A). Notopodia from segment 4, extending for 17 segments (Figs. 6A, 6C and 6D); first 8 pairs of podia long, cylindrical, following ones slender, shorter than anterior ones, almost inconspicuous. Notochaeta of two types (Figs. 7A and 7D), widely-winged, long thick chaetae in both rows throughout (Figs. 7B, 7E, 7F, 8A and 8D) and short smooth capillaries ( Fig. 7C and 8B). Neuropodia starting from segment 5, as glandular ridges slightly raised from surface of thorax until segment on which notopodia terminate, thereafter as elongate pinnules. Uncini pectinate, with 5-6 teeth, higher than long, with oblique and concave base, short triangular heel directed backwards for the attachment of the ligamental filament, with prow downwardly directed, aligned with line of teeth, connected to long filament (Figs. 7G-7J, 8C, 8E-8G). Uncini arranged in single row until segment 10, in double rows from segment 11-20, in back to back position; in single rows from segment 21 until the pygidium; abdominal uncini similar in shape but smaller in size than thoracic ones. Anus rounded, without any anal papillae (Fig. 6A). Tube with an inner lining of mucus covered by sand.
Variations. Three specimens were collected from Guangxi Province. The number of segments in these specimens varies from 125 to 130, with the highest number of segments in the holotype. One paratype was incomplete (length, 189 mm; thorax, 13.1 mm). It has ventral grooves shaped as a trapezoid on segment 13, as a square on segment 14, and as a long rectangle on segment 15. The first pair of branchiae are approximately five times larger than the subsequent ones. The other paratype is complete (length, 125 mm; thorax, 8 mm), with relatively short broken dendritic branchiae. The stems of the first pair of branchiae are slightly thicker than the subsequent ones.
Remarks. Loimia macrobranchia sp. n. is characterized by the shape and size of its first pair of large branchiae, which are approximately three to five times larger than subsequent ones, with a thick main stem and approximately 18 dendritic branches arranged in two levels. It is easy to identify Loimia macrobranchia sp. n. by its long length of 360 mm (L. borealis sp. n. 84 mm; L. ingens 5 mm; L. bandera 30 mm; L. arborea 120 mm). The largest species within this genus is Loimia ramzega Lavesque et al., 2017 from French waters (maximum length, 650 mm), and it differs from L. macrobranchia sp. n. in the shape of its first pair of branchiae, with a thick main stem and many dendritic branches arranged in five levels (Lavesque et al., 2017).
Etymology. We attribute to this taxon the epithet "macrobranchia" to indicate the first pair of large branchiae.

Phylogenetic analysis
Kimura's two-parameter pairwise genetic distance between L. arborea from this study and that from GenBank (Carr et al., 2011) was 32.1%, suggesting that they are different species. The genetic distance between Loimia sp. from India and that from Thailand was 0.6%, indicating that they are identical species. Except for Loimia sp. and L. arborea, the mean sequence divergences within species ranged from 0.0% (L. bandera and L. ramzega) to 0.4% (L. macrobranchia sp. n.), and the mean sequence divergences between species, excluding the outgroups, ranged from 15.4% (L. ingens and Loimia sp.) to 30.2% (L. arborea from GenBank and L. ingens). The highest divergence between Loimia species and the outgroups was 29.8% (L. borealis sp. n. and T. lapidaria).
The combined phylogenetic trees (Fig. 9) reconstructed from both maximum likelihoods (ML) and Bayesian inference (BI) analyses were generally comparable, and all ingroup taxa were recovered in a clade with high support value (BP (maximum likelihood bootstrap percentage) > 99%, PP (Bayesian posterior probability) = 1.0). Loimia arborea, whose sequence was downloaded from GenBank (Carr et al., 2011), clustered together with the outgroups (T. lapidaria and P. cristata) with moderately high support (BP = 73, PP = 0.99). In addition, the average genetic divergence between L. arborea species from GenBank and other L. arborea species from this study was 32.1%, indicating that they are different species. Phylogenetic tree reconstruction of the COI gene showed two well supported main clades (Fig. 9, clades A, B) with moderately high support (BP = 73, PP = 0.99). Clade A included L. ingens from China, Loimia sp. from Thailand and India, L. medusa from the USA, and L. ramzega from France. Loimia ingens and Loimia sp. formed a highly supported clade (BP = 89, PP = 1.0), indicating their close relationship. Clade B grouped L. macrobranchia sp. n., L. arborea, L. bandera, and L. borealis sp. n. (Fig. 9). Loimia arborea and L. bandera formed a strongly supported subclade (BP = 97, PP = 1.00), illustrating that they are more genetically related than the rest of the species within clade B.

DISCUSSION
We re-identified the specimens deposited in the MBMCAS under the name L. medusa and collected new specimens near their recorded sites. This analysis revealed that the specimens belonged to two different species, both new and significantly different from L. medusa as described by Hutchings & Glasby (1988). One species, L. borealis sp. n., was collected from the Shandong Peninsula. It differed from L. medusa by the presence of large round lobes on segment 3 and compact long rectangular mid-ventral shields (Table 4). The other species, L. macrobranchia sp. n., was identified in Guangxi Province. Similar to L. medusa, the first pair of branchiae were approximately three times longer than the body width. However, L. medusa had eyespots arranged in small groups on each side of the posterior prostomium, which were absent in L. macrobranchia sp. n. (Table 4). Hutchings & Glasby (1995) designated a neotype for Loimia medusa and indicated that their distribution was restricted to the Arabian Sea. Due to the long distance between China and the type locality (Red Sea, neotype from the Persian Gulf) of L. medusa, we believe that the older records of L. medusa in China are misidentifications and this species does not occur in Chinese seas. According to , Loimia montagui was initially identified as Terebella montagui from the Philippines by Grube (1878), and then assigned to the genus Loimia by Hartman (1959). Subsequently, Terebella montagui Grube, 1878 was redefined as Loimia grubei by Holthe (1986). Presently, the taxonomic significance of Loimia montagui (Grube, 1878) in WoRMS (http://www.marinespecies.org) is uncertain. Given the brief descriptions of L. grubei by Holthe (1986) and L. montagui by Hartman (1959), we referred to the descriptions of L. grubei by  and L. montagui by Caullery (1944). According to these descriptions, the species has a pair of round lateral lobes on segment 1, slightly shorter round lobes on segment 3, branchiae with short branchial filaments branching dichotomously from the secondary stems that originate from the short main stem, and ventral shields extending from segments 2 to 14 (Table 4). By contrast, L. borealis sp. n. has large ventral lateral lobes on segment 1, well-developed lobes on segment 3 that are slightly larger than those on segment 1, and seven mid-ventral shields from segments 2-10 (Figs. 2B and 2C). As for L. macrobranchia sp. n., the lobes on segment 1 are long, and they extend as elevated collars that connect mid-ventrally (Figs. 6C and 6D). In addition, 10 ventral shields from segments 2 to 12 are present. Previously, Hutchings & Glasby (1988) examined materials collected from Australia that were initially identified as L. montagui. Subsequently, they classified these specimens as L. ingens. As for the materials collected from coastal areas of Chinese seas and identified as L. ingens, they differed from L. montagui in the shape of their branchiae, namely in their long basal stems, subdistal dendritic branches, and loose spiral filaments (Fig. 5I).  suggested that the shape and position of the lobes on segments 1 and 3, the shape of the ventral glandular shields, and the number of segments on which they are present, together with the number and general morphology of the uncinial teeth, are critical in the identification of Loimia species. As observed by us, the shape and distribution of mid-ventral shields, which have rarely been included in species descriptions , are useful characteristics in the taxonomic classification of Loimia species. The mid-ventral shield shape of Loimia species varies from rectangular to trapezoidal, which is typical of Terebellidae (Nogueira, Hutchings & Fukuda, 2010;Nogueira, Fitzhugh & Hutchings, 2013). These shields usually extend from anterior segments to posterior segments where notopodia terminate, appearing blood red-to-yellowish in live materials (Figs. 1C, 5A, 5G and 6A) and pale yellow-to-white in preserved specimens (Fig. 5B). Londoño-Mesa (2006, 2009) and Londoño-Mesa & Carrera-Parra (2005) commented on the usefulness of the distribution of ventral shields as a specific characteristic for Loimia species, although it is difficult to determine the exact anterior segment (mostly short and fuzed to each other) on which the shields begin, and the particular segment on which they end.  also mentioned that differences in the shape of shields was an additional useful characteristic in the identification Loimia species.
After re-identifying the specimens that have been deposited in the MBMCAS for many decades, we found that the important morphological characteristics of branchiae and lobes were destroyed, and their shape was undefinable (Figs. 2D-2F, 6F and 6G). By contrast, the characteristics of mid-ventral shields were well preserved, and they were easily recognizable. Equally-sized mid-ventral shields in L. borealis sp. n. spanned from segments 2 to 10, each with a length significantly longer than the width (Figs. 2D-2F). In old materials of L. macrobranchia sp. n. and L. ingens, the rectangular ventral shields spanned from segments 2 to 12 (Figs. 6F and 6G), similar to the shields in new materials. In agreement with , we conclude that the shape of mid-ventral shields is a significant character for the identification of species of Loimia.
In the phylogenetic tree ( Fig. 9), Loimia macrobranchia sp. n. was recovered as a monophyletic clade, although nodal support was low. Loimia borealis sp. n. was retrieved as a sister to the clades of L. arborea and L. bandera with moderate support (Fig. 9). In addition, Kimura's two-parameter pairwise genetic distance between L. macrobranchia sp. n. and other Loimia species ranged from 18% to 24%, and the genetic distance between L. borealis sp. n. and other species ranged from 20% to 24%, indicating genetic divergences between new species and other Loimia species.

CONCLUSIONS
This study is the first to analyze molecular sequence data of Loimia species from Chinese seas. The new species L. borealis sp. n. and L. macrobranchia sp. n. were established based on their morphological and molecular findings. Loimia borealis sp. n. is distinguished from other valid Loimia species by its compact, slender, and rectangular ventral shields, which indicates that the ventral shield shape is a significant characteristic in the identification of Loimia species. The other species, L. macrobranchia sp. n., is characterized by its large size and large first pair of branchiae. A revision of the genus Loimia from Chinese seas was made, and we suspect that older records of L. medusa and L. montagui in China are misidentifications. A key for the identification of Loimia species in Chinese seas is provided. This key only contains valid species listed in WoRMS (http://www.marinespecies.org).
Key to the species of Loimia found in China seas