Demystifying the Capitella capitata complex (Annelida, Capitellidae) diversity by morphological and molecular data along the Brazilian coast

The sibling species of Capitella capitata are globally known for their tolerance to disturbed habitats and the C. capitata complex is often used as an ecological indicator. A recent re-description proposed that C. capitata, originally described in Greenland is restricted to the Artic and Subarctic regions. Given their ecological relevance, we conducted a morphological and molecular analyses based on mtDNA sequences to investigate the diversity and distribution of the C. capitata complex along the Brazilian coast. Our morphological and molecular data were congruent and revealed the existence of four new species distinct from C. capitata, collected from the type locality. This study is the first characterization of the biodiversity and distribution of Capitella species made along the Brazilian coast and yielded a set of morphological characters corroborated by the mtDNA sequences for species identification. Our results increase the biodiversity of the genus along the Brazilian coast by describing four new species (Capitella aracaensis sp. n., Capitella biota sp. n., Capitella neoaciculata sp. n. and Capitella nonatoi sp. n.). One species was collected from only one sampling site, while the others are distributed along the coast.


Introduction
The genus Capitella Blainville 1828 [1] (Capitellidae, Annelida) comprises polychaetes characterized by nine thoracic chaetigers with a species variation associated with the number of chaetigers bearing capillary chaetae and hooded hooks, genital spines present in chaetigers 8-9 of males and hermaphrodites, and the pygidium lacking appendages. There are 19 nominal species of Capitella described in a variety of environments (e.g., intertidal zone to abyssal depths) and habitats (e.g., soft-bottom, egg capsules, whale-bones) worldwide [2][3][4][5]. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 General attributes traditionally used to characterize capitellid species include overall size, shape and relative size of the prostomium and peristomium, formation of the peristomium as a complete or an incomplete ring, the number and distribution of capillary chaetae and hooded hooks along the thorax, the morphology of the genital spines, the number, size and structure of the hooded hooks and the shape of the pygidium [2][3][4][5][6]. However, distinguishing Capitella species is difficult and includes multiple complexes of cryptic species, which morphology alone has failed to define since the morphological features and life history traits can overlap.
The genus Capitella was the first marine invertebrate genus identified as a complex of cryptic species through molecular markers (allozyme) in the pioneer work of Grassle & Grassle [7], in which six different species, previously considered as Capitella capitata, were discovered along a short stretch of the USA Atlantic coast. Subsequent studies also identified cryptic species of Capitella through differences in their reproductive modes [8][9], gamete and larvae ultrastructure [10][11], developmental rates, dispersal patterns [12][13][14], adult body sizes [15] and physiological characteristics [16][17]. These criteria were used to define at least twelve cryptic species that were previously identified in laboratory cultures [3].
Capitella capitata [18] was originally described in Greenland, but the reports of its occurrence were later expanded to all oceans [3,[19][20][21]. The poor taxonomic understanding of the complex of cryptic species has led to broad distributions of these taxa, also known as 'the cosmopolitan syndrome' [22][23]. However, careful morphological revision of specimens and the advent of molecular techniques have increased the local diversity of several polychaete species and diminished the geographical distribution of species previously considered to be cosmopolitan [24][25][26][27][28][29].
After his re-description of C. capitata from the type locality, Blake [3] proposed this species is restricted to the Artic and Subarctic regions. Specimens of C. capitata that were recorded along the Brazilian coast [30][31] were therefor apparently misidentified. Moreover, the records of C. capitata from diverse benthic habitats and depths throughout the world indicate that a number of sibling and undescribed species are being overlooked taxonomically and ecologically.
Capitella capitata is referred to as an important ecological indicator, due to its high densities in polluted ecosystems [32][33] and as a model organism in many ecotoxicological studies [17,[34][35]. However, ecological studies are likely referring to an unknown number of species with different local and regional distributional patterns, toxicity tolerance [17], and reproductive strategies [34]. Given the ecological importance of this group the correct delimitation of species is essential. The aims of the present study were to investigate the diversity and distribution of the Brazilian populations of the C. capitata complex and characterize them morphologically and molecularly. Morphological characters and mtDNA sequences (COI and 16S) of specimens from 13 sites along the Brazilian coast were analyzed. Sequences from the type locality and public datasets were also included. The results will be useful in order to correctly identify the species in further distinct studies.

Study areas and sampling
Samples were collected from the intertidal zone and shallow waters (up to 0.5 m deep) at 13 sites along the Brazilian coast in six different states (Fig 1). Samples were also collected at one site in Greenland (02-Aug-2013; 69.25˚S 54.10˚W). Collected sediments were washed in the field on a 500 μm and 300 μm mesh sieve and any Capitella worms retained in the residue were fixed and preserved in 92% ethanol.
The nomenclature used for chaetal morphology follows that usually used for Capitellidae species and some suggested by Green [2]. The chaetal characters used here are: • main fang: format and angle with the hook shaft; • teeth: number of teeth and rows that are arranged above the main fang; • shoulder: development of the curvature of the hook; • hood: size, format, size of the opening and texture; • anterior shaft: length between shoulder and node; • node: presence or absence and shape; • posterior shaft: length between node and the end of the hook.
The material was deposited at the Museu de Zoologia, Universidade Estadual de Campinas (ZUEC), São Paulo, Brazil and the Museu Nacional do Rio de Janeiro (MNRJ), Rio de Janeiro, Brazil.

Nomenclatural acts
The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) 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: 7C72C6E6-F616-4A60-87D7-C2239F03AB14. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS.

Molecular analysis
DNA extraction, amplification and sequencing. Genomic DNA was extracted with a DNeasy Blood & Tissue Kit (QIAGEN) and in some cases according to the protocol in Floyd et al. [36]. Fragments of the mitochondrial gene, cytochrome oxidase subunit 1 (COI), were amplified using universal primers LCO1490 and HCO2198 [37] or degenerated primers degLCO1490 and degHCO2198 [38]. For the 16S ribosomal DNA (16S) the universal primers 16Sar-L and 16Sbr-H [39] were used. PCR reactions for both loci consisted of PuReTaq Ready-To-Go™ PCR Beads (GE Healthcare), 1.5 μL of each primer (0.6 mM), 2 μL of DNA and 20 μL of water. The thermal cycling conditions for COI were one cycle of 94˚C for 3 min, 5 cycles of 94˚C for 40 s, 47˚C for 40 s and 72˚C for 1 min, 32 cycles of 94˚C for 40 s, 52˚C for 40 s and 72˚C for 1 min, followed by a final extension step of 72˚C for 5 min. For the 16S amplification, the reaction consisted of one cycle of 94˚C for 3 min, 5 cycles of 94˚C for 30 s, 42˚C for 40 s and 72˚C for 90 s, 32 cycles of 94˚C for 30 s, 46˚C for 40 s and 72˚C for 90 s, followed by a final extension step of 72˚C for 7 min. The resulting PCR products were purified and sequenced by Macrogen, Inc.
Data analysis. Electropherograms were edited with Sequencher 4.1 (Gene Codes Corporation), and sequences were aligned with MAFFT 7.0 [40] using the G-INS-I strategy for COI and Q-INS-I for 16S. COI sequences were checked for translation using the invertebrate mitochondrial genetic code. As saturation was not detected in the saturation test [41], all codon positions were used, as implemented in DAMBE5 [42]. The phylogenetic analysis was performed using the maximum likelihood (ML) algorithm and Bayesian inference (BI). Three datasets were considered, COI, 16S and concatenated (COI+16S). The ML trees were estimated in RAxML 8.2 [43] using the substitution models GTR+G for all datasets. The best ML tree was obtained from 20 initial independent trees, and the statistical support was obtained with a rapid bootstrap function (-f a) using 1000 replicates. The BI was conducted in MrBayes 3.2 [44] under the HKY +I+G model for COI and 16S, and HKY+G for the concatenated dataset, as selected in the jMo-delTest 2 [45] using Bayesian information criterion (BIC). Tree parameters were sampled every 1000 generations, for a total of 10 7 samples. Two independent runs and 4 chains were implemented. The final results were checked according to the standard deviations of split frequencies (<0.01). The effective sampling size (ESS>200) was assessed via Tracer 1.5 [46]. The ML and BI analyses were conducted in CIPRES Science Gateway [47]. Species from two other Capitellidae genera, Notomastus (16S and COI) and Heteromastus (COI), were considered as outgroups and were used to root the trees. The concatenated dataset used only Notomastus as an outgroup as no 16S sequences are available for Heteromastus. Intra-and interspecific pair-wise genetic distances were estimated under the p-distance and Kimura 2 parameters models with MEGA 6.1 [48]. GenBank sequences of C. capitata from other localities were taken to compare with our sequences. Data collection of specimens, museum codes and GenBank (www.ncbi.nlm.nih.gov/ genbank) accession numbers are detailed in Table 1.

Morphological analysis
Examination of 4,423 specimens from different localities along the Brazilian coast allowed us to classify them into four species within Capitella: 33 C. aracaensis sp. n., 149 C. biota sp. n., 734 C. neoaciculata sp. n., and 3,507 C. nonatoi sp. n. These species were identified mainly by the overall size, shape and size of the prostomium and peristomium, formation of the peristomium as a complete or an incomplete ring, the number and distribution of capillary chaetae and hooded hooks along the thoracic chaetigers, the details of the genital spines, the number, size and structure of the hooded hooks and the shape of the pygidium. The differences of these characteristics among the Capitella species were already summarized in a table by Silva et al. [5]. Here, we provide the description of the species and a key to the new species among all valid Capitella species.
Type locality. West Greenland.
6d. Hooded hooks on chaetigers 8 and 9; prostomium short and rounded with a mid-ventral depression; peristomium forming an incomplete achaetous ring; eyespots absent; hooded hooks with numerous teeth above main fang arranged in five rows: C. capitata (Fabricius 1780) [18] 6e. Hooded hooks on chaetigers 8 and 9; prostomium conical with a dorsal depression and a ventral groove; peristomium forming a complete achaetous ring; eyespots present in juveniles; hooded hooks with numerous teeth above main fang arranged in four rows: C. caribaeorum Warren & George 1986 [60] 6f. Hooded hooks on chaetigers 8 and 9; prostomium conical; peristomium forming an incomplete achaetous ring; eyespots absent; hooded hooks with four teeth above main fang in a single row: C. dizonata Johnson 1901 [61] 6g. Hooded hooks on chaetigers 8 and 9; prostomium rounded; peristomium forming a complete achaetous ring; eyespots absent; thoracic hooded hooks with 5 − 6 teeth above main fang arranged in two rows; abdominal hooded hooks with 8 − 9 teeth above main fang arranged in three rows: C. iatapiuna Silva et al. 2016 [5] 6h. Hooded hooks on chaetigers 8 and 9; prostomium rounded with a dorsal smooth depression and ventral groove; peristomium forming an incomplete achaetous ring; eyespots present; thoracic hooded hooks with six teeth above main fang arranged in two rows; abdominal hooded hooks with three teeth above main fang arranged in two rows: C. nonatoi sp. n.
Capitella Additional material examined (S1 Appendix): São Paulo, Araçá Bay (25 specs.). Description. Based on type material, additional material and specimens examined by SEM. Size range of material examined (complete individuals) 11.37-15.44 mm long, 0.45-0.6 mm wide and 50-62 chaetigers. Specimens slightly widest anteriorly, gradual narrowing posteriorly. Color in alcohol brownish. Prostomium conical, wider than longer (Figs 2A and 2C; 3A and 3D). Peristomium large, forming a complete achaetous ring, conspicuous dorsal and ventrally, wider than peristomium (Figs 2A and 2B; 3A and 3B); eyespots absent. Nuchal organs not visible using light microscopy or SEM. Chaetigers 1-4 similar, rectangular, weakly biannulate; chaetigers 5-9 similar, square, with mid-ventral and lateral groove (Figs 2C and 2D; 3D and 3E; 4A and 4B). Male and female adult specimens with unilimbate capillaries in notopodia and neuropodia of chaetigers 1-7, hooded hooks in neuropodia of chaetigers 8-9 and genital spines in notopodia of chaetigers 8-9. Notosetae arranged in a single row of 3-7 capillaries and 5-6 hooded hooks; neurosetae arranged in a single row of 3-8 capillaries and 4-6 hooded hooks. Thoracic hooded hooks with pointed, straight and short main fang, right angle with the shaft, surmounted by 2 apical teeth, one above the other; long curved shoulder; anterior shaft absent; developed node; long and slightly curved posterior shaft; short and smooth hood ( Fig  2F). Chaetigers 8 and 9 with two straight genital spines with tips sharply curved and thin vertical grooves; spines of chaetiger 8 embedded and shorter than those of chaetiger 9; spines of chaetiger 9 external and larger than those of chaetiger 8 (Figs 2E; 3F and 4D). Division between thorax and abdomen not prominent (Figs 2C and 2D; 3D and 3E; 3A and 3B). Abdominal chaetigers as long as wide (Figs 2C and 3D); chaetigers with 8-9 hooded hooks in notopodia and 9-11 in neuropodia, reduced to one hook in far posterior; hooks slightly smaller than the thoracics with pointed, straight and short main fang, right angle with the shaft, protruding just slightly through frontal opening, surmounted by 2 teeth, one above the other; long and curved shoulder; anterior shaft absent; well-developed node; long and curved posterior shaft; long and smooth hood (Figs 2G; 3G and 4E). Branchiae absent. Pygidium a quite small simple lobe without anal cirri (Figs 2H and 3H). Methyl green staining pattern. Chaetigers 5-7 with a strip of small spots in the middle of the segment, chaetigers 8 and 9 darkly stained and abdominal segments staining uniformly (Fig 3C).
Biology. All specimens with genital spines. However, a few specimens presented oocytes inside the abdominal region, confirming they are female specimens.  Remarks. Capitella aracaensis sp. n., belongs to a group of species of Capitella with capillary chaetae on chaetigers 1-7 and hooded hooks on chaetigers 8 and 9. This group includes C. amboensis, C. capitata, C. caribaeorum, C. dizonata, C. iatapiuna, C. perarmata, C. singularis and C. teleta. Capitella aracaensis sp. n. shares some features with C. caribaeorum, C. iatapiuna and C. singularis, such as the peristomium forming a complete ring and the absence of eyespots. These species differ, however, in the characteristics of their prostomium: in C. caribaeorum it is conical, with a dorsal depression and ventral groove; in C. iatapiuna it is quite rounded, as long as wide; and in C. singularis it is conical and smooth; while in C. aracaensis sp. n. it is rounded. The abdominal hooded hooks also differ in number and distribution of teeth above main fang: in C. caribaeorum there are several teeth arranged in four rows; in C. iatapiuna there are 5 − 6 teeth arranged in two rows on thoracic hooks and 8 − 9 teeth in three rows on abdominal hooks; and in C. singularis there are 11 − 14 teeth arranged in two rows; while in C. aracaensis sp. n. there are two teeth, one above the other, on both thoracic and abdominal hooded hooks. Capitella capitata, C. dizonata, C. perarmata and C. teleta differ from C. aracaensis sp. n. by having a peristomium forming an incomplete ring and in features of the hooded hooks. Capitella aracaensis sp. n. can be distinguished by its rounded prostomium, large complete peristomium and hooded hooks with two teeth above the main fang, one above the other.
Etymology. This species was named after the Araçá Bay (São Sebastião, state of São), which has a high biodiversity [64] and is one of the samplings areas of this study.
Habitat. Intertidal region, in fine sand and mangrove.   Prostomium pointed, longer than wider (Figs 5A and 5B; 6A and 6B). Peristomium distinct, forming a complete achaetous ring, conspicuous dorsal-and ventrally, wider than prostomium (Figs 5A and 5B; 6A and 6B); eyespots absent. Chaetigers 1-7 similar; chaetigers 8 and 9 slightly more narrow; all chaetigers with shallow intersegmental furrows; chaetigers 7-9 with a deep mid-ventral groove (Figs 5D and 6E) and a smooth lateral groove (Fig 6D). Adult specimens with unilimbate capillaries in notopodia and neuropodia of chaetigers 1-3 and hooded hooks in notopodia and neuropodia of chaetigers 4-9. Notosetae arranged in a single row of 3-7 capillaries and 7-8 hooded hooks; neurosetae arranged in a single row of 3-8 capillaries and 5-9 hooded hooks; chaetae emerging from the middle of the chaetigers (Figs 5D and 6D). Thoracic hooded hooks with pointed and short main fang, upward curved, surmounted by one apical tooth; long and slightly curved shoulder; long and smooth hood (Fig 6F). Division between thorax and abdomen not prominent (Figs 5D; 6D and 6E). Abdominal chaetigers as long as wide, anterior chaetigers with 6-9 hooded hooks in notopodia and 6-10 in neuropodia, reducing to 2-3 hooks in posterior chaetigers, emerging from the last third of the chaetigers. Hooded hooks small with rounded and robust main fang, right angle with the shaft, protruding through the frontal opening, surmounted by two teeth, one above the other; long and slightly curved shoulder; anterior shaft absent; developed node; long and well curved posterior shaft; long and smooth hood (Figs 5F; 6H). Chaetigers 8 and 9 with 2 embedded genital spines. Spines of chaetiger 8 thin, straight and with slightly curved tips; spines of chaetiger 9 larger than those of chaetiger 8, straight and with slightly curved tips (Figs 5E; 6G). Branchiae absent. Pygidium small simple lobe without anal cirri (Fig 6I).
Remarks. Capitella biota sp. n., belongs to a group of species of Capitella with capillary chaetae on chaetigers 1-3. This group includes C. capitata tripartita, C. jonesi and C. minima tulearensis. Although C. jonesi and C. minima tulearensis also have hooded hooks in noto-and neuropodia of chaetigers 4 − 9, the former has a peristomium forming an incomplete ring and eyespots, while the latter has hooded hooks with 7 − 9 teeth above main fang, distributed in three rows, both differing from C. biota sp. n. Capitella capitata tripartita differs from C. biota sp. n. in having eyespots and mixed capillary chaetae and hooded hooks in noto-and neuropodia of chaetigers 4-9. Capitella biota sp. n. can be distinguished by having a peristomium forming a complete ring and thoracic hooded hooks with one apical tooth and abdominal hooks with two teeth above main fang, one above the other.
Etymology. This species was named after the "BIOTA-FAPESP Program", which allowed the realization of the "BIOTA-Araçá Project", responsible for funding the collection of most of the individuals reported in this paper.
Habitat. Intertidal region, in fine sand and mangrove sediments. . Noto-and neuroaciculae arranged in a single row of 2 or 3 acicular spines; noto-and neurosetae arranged in a single row of 5 − 13 unilimbate capillaries and 6 − 10 hooded hooks; chaetae emerging from the last third of the chaetiger. Thoracic hooded hooks with a rounded, thick and slightly curved main fang, surmounted by six apical teeth arranged in two rows (3 basally and 3 in superior row); long straight shoulder; short anterior shaft; inconspicuous node; long posterior shaft; long and smooth hood (Figs 7E and 7F; 8F and 9F). In females, chaetiger 9 with a pair of small, thin and slightly curved genital spines. In males, chaetiger 8 with 6 falcate external genital spines (2 fascicles) with tips sharply curved, narrower than those of chaetiger 9; chaetiger 9 with 6 straight embedded genital spines (2 fascicles), curved apically, with blunt tips, larger than those of chaetiger 8 (Figs 8J and 9C). Division between thorax and abdomen prominent. Abdominal chaetigers as long as wide; chaetigers with 3 − 10 hooded hooks in notopodia and 5 − 15 in neuropodia, reducing to 4 notopodial and 7 neuropodial hooks. Abdominal hooded hooks shorter than the thoracics with a pointed, thin and slightly curved main fang, surmounted by six teeth arranged in two rows (3 basally and 3 on superior row); long and slightly curved shoulder, short anterior shaft; developed node; long posterior shaft; short and smooth hood (Figs 7G and 7H; 8G, 8H and 8I and 9G). Ventral depression along the abdominal region (Figs 8K and 9H). Neuropodial hooded hooks emerging from long parapodial ridges in far posterior chaetigers (Figs 8K and 9H). Branchiae absent. Pygidium a large simple lobe, fused dorsally and slightly bilobate ventrally (heart-shaped), without anal cirri (Figs 7I and 7J; 8K and 9H).
Methyl green staining pattern. Specimens staining uniformly, except the two first chaetigers which are lightly speckled (Fig 8C).
Variation. The presence of the acicular spines varies according to the length and perhaps sexual development of the specimens. Large females (more than 0.8 mm wide) can have acicular spines in notopodia of chaetiger 1, however the aciculae are less developed than in males; and small males, with genital spines less developed, commonly lack acicular spines in neuropodia of chaetigers 1 and 2. Larger specimens can also have capillary chaetae on chaetiger 9.
Biology. Almost all specimens with genital spines; specimens with small and less developed genital spines only on chaetiger 9 presented oocytes inside the abdominal region, confirming they are female specimens. Males, with robust genital spines, did not present oocytes.
Remarks. Capitella neoaciculata sp. n., belongs to a group of species of Capitella with capillary chaetae on chaetigers 1-8 and hooded hooks on chaetiger 9. This group includes C. aciculata and C. teres. Capitella neoaciculata sp. n. differs from C. teres by having acicular spines in Diversity of Capitella along the Brazilian coast than five teeth (3 basally and 2 on superior row), there is a ventral depression along the abdominal region, and the pygidium is large and heart-shaped rather than inconspicuous and a simple ring as in C. aciculata.
Etymology. This species was named based on the presence of acicular spines as Capitella aciculata, however, with a new combination of characters.
Methyl green staining pattern. Chaetigers 5-8 darkly stained, chaetigers 8 and 9 and the first two abdominal with dark speckles (Fig 11C). Diversity of Capitella along the Brazilian coast Biology. All specimens without genital spines, with oocytes in the abdominal region confirming they are female specimens.
Remarks. Capitella nonatoi sp. n. belongs to a group of species of Capitella with capillary chaetae on chaetigers 1-7 and hooded hooks on chaetigers 8 and 9. Capitella teleta resembles C. nonatoi sp. n. in the overall body shape, peristomium forming an incomplete ring, a very conspicuous nuchal organ and presence of eyespots. However, the former differs from C. nonatoi sp. n. in having a flattened and long prostomium, while in C. nonatoi sp. n. the prostomium is short and rounded. The differences are also related to the number and features of the abdominal hooded hooks and number of genital spines. Capitella teleta has 4 − 6 hooks in notopodia and 5 − 6 in neuropodia, with six teeth above main fang, while C. nonatoi sp. n. has 2 − 5 hooded hooks in notopodia and 4 − 10 in neuropodia, with three teeth arranged in two rows. Furthermore, C. teleta has 6 − 8 genital spines on chaetiger 8 and C. nonatoi sp. n. has four. Capitella caribaeorum, C. iatapiuna and C. singularis differ from C. nonatoi sp. n. by having a peristomium forming a complete ring and the absence of eyespots. Besides C. perarmata, C. capitata and C. dizonata also have a peristomium forming an incomplete ring but they differ from C. nonatoi sp. n. by the absence of eyespots and features of the hooded hooks such as number of teeth and their distribution above main fang. Capitella nonatoi sp. n. can be distinguished by its short and rounded prostomium with a dorsal smooth depression and a ventral groove, and eyespots.
Etymology. This species was named in memorian of Professor Edmundo Ferraz Nonato, who was the pioneer on taxonomy of Polychaeta in Brazil and who had influenced many researchers with his dedication, knowledge and passion for polychaetes and science.
Habitat. Intertidal region, in fine sand and mangrove sediments.

Molecular analysis
The phylogenetic analyses were based on 134 sequences of 16S and 106 of COI. The final alignment consisted of 558 bp for 16S and 655 bp for COI. Excluding the outgroup species, the 16S sequence had 215 (38.5%) variable sites, while the COI sequence had 273 (41.7%). The maximum likelihood and Bayesian inference method recovered the same species, as had been previously identified here by morphological characteristics (C. aracaensis sp. n., C. biota sp. n., C. neoaciculata sp. n. and C. nonatoi sp. n.). The four species clades were reciprocally monophyletic and were well supported in all datasets (COI, 16S and concatenated) and recovery methods (Fig 13 and S1 Fig). The Capitella nonatoi sp. n. clade included individuals from 12 sampling sites, while C. neoaciculata sp. n. was present in 6 sites, C. biota sp. n. in two sites and C. aracaensis sp. n. in only one site.

Discussion
The use of mtDNA sequences for species identification may speed up global diversity estimations [65], while the robustness of the morphological information improves the quantity and quality of species descriptions, as well as the understanding of the morphological adaptations in their evolutionary history [66][67]. Our morphological and molecular data were congruent and supported the existence of four different species of Capitella, all of them rather distinct from Capitella capitata, increasing the biodiversity of the genus along the Brazilian coast.
The delimitation of the boundaries of the various Capitella species along Brazilian waters has revealed a hidden local diversity and may enhance assessments of environmental health [68] as an indicator of organic pollution [69][70][71][72]. Sympatric occurrences were observed in six sites; most species distributed along the coast, while one only at only one site (Capitella aracaensis sp. n.). Combined morphological and molecular approaches were also conducted and have yielded similar results [25][26][73][74]. Indeed, these authors identified distinct species among the species complexes, increasing the richness of the target genus [25,26,73,74]. Interestingly, the taxonomic confusion surrounding Capitella species can be illustrated through a number of studies that focused on the genus, but were unable to identify specimens at the species level [75][76][77][78][79].
Our results indicate that many studies may have overlooked the richness of Capitella species due to the challenges in the morphological taxonomic identification, despite the ecological importance of the genus [7]. The four new species described here were morphologically differentiated from all congeners based mainly on the shape of the prostomium, peristomium and thorax, as well as the shape and number of thoracic and abdominal hooded hooks, the shape of the pygidium and of the genital hooks. The differences of these characteristics among the Capitella species are summarized in a table in [5]. Although the morphology of the prostomium and peristomium may be affected by distinct fixation protocols [80], they were very informative characteristics and should be explored, as they are quite variable and diagnostic for species within the genus [3][4][5][6]59]. We would like to highlight the fact that these new species were distinguished initially by a morphological analysis. The sequence data just confirmed our findings and helped to support the specific status of the recognized morphospecies.
Among these new species, the most widespread and abundant was C. nonatoi sp. n., with records in all sampled sites along 4,500 Km along the Brazilian coast, from the North (Pará) to the South (Paraná). Due to its high abundance and wide distribution, this species has likely been misidentified as C. capitata by previous studies [81][82][83][84]. The second most widespread species was C. biota sp. n., which was reported from the Northeast (Bahia) to the Southeast (São Paulo), ranging over an area of 1,630 Km. Capitella aracaensis sp. n. was more rare in our sampling, with low abundances and was restricted to one site of the Southeast Brazil (São Paulo). We suggested that this species is rare because Araçá Bay [64], the type locality, was exhaustively and systematically sampled over a large area during the four years of the BIOTA-FAPESP Program (Thematic Grants, Process 2011/50317-5). During these four years we found just a few specimens of Capitella aracaensis sp. n., meanwhile the other three species were frequent and abundant at the same site. The discrepancies regarding the geographic distributions of these species could be explained by sampling effort, biological aspects such as their differing reproduction strategies [85], larvae development, colonization capacity [86], and physiological tolerances [17].
The scale bar represents the average nucleotide substitutions per site. Brown arrow shows the Capitella capitata from the typelocality (west Greenland). Gray boxes indicate Genbank sequences from Capitella species. Asterisks (*) indicates that the support value was lower than 70 (ML) or 0.7 (BI), and a dash (-) indicates that the branch was not recovered.
https://doi.org/10.1371/journal.pone.0177760.g013 Table 2. Intraspecific (in bold) and interspecific mean pairwise genetic distance based on Kimura-2-parameters (K2P) for 16S (on top) and COI (below). All values are in percentage. Number in parenthesis represents the standard error. The low levels of intraspecific molecular variations recorded for all the four Capitella species (with a maximum of 2.2% for C. biota sp. n.) were expected within the same lineage. Usually values lower than 6% indicate that lineages are not composed of some sub-complex of the species [74]. On the other hand, high interspecific variation, as was observed here, 15.7% up to 26.5% for COI, and 24.4% up to 46.4% for 16S was observed in other congeners, including between the deep-sea species, for example C. iatapiuna and C. teleta (20% for 16S) [5]. A similar molecular interspecific variation was found for errant [24,87], and sedentary annelids [27].
The COI values were compatible with the 10-fold rule of species delimitation [88], supporting the hypothesis that the sample groups indeed represent four different species. Therefore, no less conservative method of species delimitation based on cluster recognition [89] was necessary to confirm this status. Recently, COI sequences have been applied to clarify the systematics of the Capitella capitata complex [90][91]. A similar result was found in the Gulf of Mexico, with two species under the name C. capitata, with 21.7% of K2P divergence [90], while [91] found the same haplotype of C. teleta both on the Atlantic coast of USA and Japan. Despite the fact that these two studies are inconclusive regarding their morphological approaches, intermediate morphologies between the species [90] and an intraspecific variability within the species [91] were found.
Thus, considering the intra and inter-specific variation calculated for these particular mitochondrial genes, we concluded that these divergences support the distinction among the four morphologically delimitated species. Furthermore, a genetic distance comparison showed that the COI values were higher than 16S values when considering intraspecific comparisons. However, in relation to the differences between species, the COI values were smaller than the 16S values.
In addition to morphological and molecular studies confirming the cosmopolitan status of some species [92][93], our findings support restricting the geographical range of C. capitata, as was previously proposed by Blake [3]. Furthermore, we revealed a hidden diversity within this genus along the Brazilian coast. Despite the fragmentation of the C. capitata complex in at least four species, this does not indicate that each species has a restricted distribution, as frequently expected in this type of study. Thus, even for marine invertebrates with a supposedly low potential for dispersion, the population cohesion along a wide distribution can be maintained.
Finally, this study showed the importance of exhaustive and systematic sampling to unveil cryptic and rare species. Furthermore, this result emphasized the effectiveness of careful and accurate morphological study as a tool to differentiate species of Capitella. The number in nodes represent the support values for ML (bootstrap-on top) and BI (posterior probability-below). The BI trees were not represented. The scale bar represents the average nucleotide substitutions per site. Brown arrow shows the Capitella capitata from typelocality (west Greenland). One asterisk ( Ã ) indicates that only COI sequence was used and two asterisks ( ÃÃ ) indicate that only 16S sequence was used. A tilde (~) indicates that the support value was lower than 70 (ML) or 0.7 (BI), and a dash (-) indicates that the branch was not recovered. (TIF) S1 Table. Genetic distance. Intraspecific (in bold) and interspecific mean pairwise genetic distances based on p-distance for 16S (on top) and COI (below). All values are in percentage. The number in parenthesis represents the standard error. (DOCX)