The Multitentaculate Cirratulidae of the Genera Cirriformia and Timarete (Annelida: Polychaeta) from Shallow Waters of Brazil

A large number multitentaculate cirratulids have been described worldwide but most are only known through the original descriptions. Type material, voucher and recently collected specimens from Brazil were revisited in order to reveal their true identity and confirm the records of widely distributed species in this region. Six species are described, three of which are new, Cirriformia capixabensis sp. nov., Cirriformia chicoi sp. nov. and Timarete ceciliae sp. nov. COI and 16S sequences were obtained and used for inter-specific comparisons. Timarete caribous is reported from several localities along the Brazilian coast and a new synonym, Cirratulus melanacanthus, is proposed. The species Timarete oculata, originally described from Brazil and lumped into the Timarete filigera species complex, is herein revalidated and redescribed. The occurrence of the species Timarete filigera and Cirriformia tentaculata is not confirmed from the Brazilian coast. Descriptions, illustrations and a key to genera and species are provided.


Introduction
The multitentaculate cirratulids form a clade composed of five genera, Cirratulus Lamarck, 1801, Timarete Kinberg, 1866, Protocirrineris Czerniavsky, 1881, Cirriformia Hartman, 1936, and Fauvelicirratulus Ç inar & Petersen, 2011. All of these genera possess more than a single pair of tentacles attached on the dorsal part of anterior segments. These dorsal tentacles are either organized in longitudinal rows as in Protocirrineris species or in transverse or oblique groups in the remaining genera. The number, segmental origin and distribution of branchial filaments are also of generic interest. Timarete species usually have branchiae originating from the posterior end of the peristomium, anterior to the dorsal tentacles and these branchiae shift to a more dorsal location, posteriorly. The branchial filaments in Cirriformia species remain near the notopodial lobes throughout the body. Branchiae in species of Cirratulus begin on the same segment as the tentacular filaments, usually chaetiger 1. The recently described genus Fauvelicirratulus has a similar arrangement of branchiae and tentacles as in Cirratulus but differs by the presence of more than a single pair of branchiae per segment and by different types of spinous chaetae.
Most of the recent literature on multitentaculate cirratulids from Brazil has referenced Timarete filigera and Cirriformia tentaculata (Also referred to as Timarete tentaculata) [8] but these are Mediterranean and northern Atlantic species and unless it was a human-mediated introduction, it is unlikely to occur in South America. For this reason, we investigate herein the identity of multitentaculate Cirratulidae from Brazil through re-examination of types, voucher material and recently collected specimens along the Brazilian coast. This study is the first attempt to review the multitentaculate cirratulids from the Brazilian coast and serves as a baseline for future studies concerning this diverse and complex group.

Materials and Methods
Specimens from several localities along the Brazilian coast ( Fig. 1) were collected in soft sediment, macroalgae and coralline algae along the intertidal zones; sediment was collected with a shovel and sieved through a 0.5 mm screen. Specimens collected in Salvador, northern Brazil ( Fig. 1) were observed and photographed live after anesthetization in 8% MgCl 2 for a few minutes. Most of the specimens used for morphological analyses were fixed in 5% formalin for at least 24 hours and transferred to 70% ethanol. Molecular analyses were carried out with specimens preserved in 92.8% ethanol.
Samples from São Pedro e São Paulo Archipelago were collected in the subtidal zone by SCUBA or free diving. A collecting permit for the marine protected areas (i.e. São Pedro e São Paulo Archipelago, Fernando de Noronha and Rocas Atoll) was granted by the Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio) under permit number 23760-2 to P.C. Paiva. Sampling in unprotected areas (including outside the National Park of Abrolhos Atoll) were under permit number 10238-1, also granted by ICMBio to P.C. Paiva.
Type material and voucher specimens of cirratulids described or reported from South America were borrowed from the United States National Museum of Natural History, Smithsonian Institution, Washington, D. C., USA (USNM) and Museum für Naturkunde, Berlin, Germany (MfN). Holotypes and paratypes are deposited at the Polychaete Collection ''Edmundo Ferraz Nonato'' -Instituto de Biologia, Universidade Federal do Rio de Janeiro (IBUFRJ).
Specimens were examined using a phase contrast light microscope and some with a Scanning Electron Microscope (SEM). Line drawings were made with a camera lucida attached to a compound microscope and length and width measurements were taken with an ocular micrometer that was calibrated with a stage micrometer. SEM preparations included dehydration through a series of increasing concentrations of ethanol ending with two changes of absolute ethanol followed by critical point drying (in a SAMDRI-795). Worms were mounted on stubs and coated with gold/palladium for two minutes at 5 nm thickness. SEM observations were carried out using the Hitachi S-4800 at the Biological Electron Microscopy Facility (BEMF), University of Hawaii at Manoa.
Genomic DNA was extracted with a Puregene Core Kit A (Gentra Systems) or according to the protocol developed for nematodes by [9]. Fragment sequences of two mitochondrial markers were obtained via polymerase chain reaction (PCR). Cytochrome oxidase subunit I (COI) was amplified using universal primers, LCO1490 and HCO2198 [10]. Each 25 mL reaction included 1X Taq buffer, 3.0 mM of magnesium chloride, 1 mg/ ml of Bovine Serum Albumin (BSA), 0.2 mM of dNTPs, 0.6 mM of each primer, 1 unit Taq DNA polymerase and 1 mL of extracted DNA. PCR conditions were one cycle of 94uC for 2 min, five cycles of 95uC for 40 s, 45uC for 90 s and 72uC for 1 min, followed by 35 cycles of 95uC for 40 s, 51uC for 45 s and 72uC for 1 min, and a final extension step of 72uC for 5 min. 16S ribosomal RNA (16S) was amplified using a forward primer 16Sar-L [11] and a reverse primer 16SAN-R [12]. Each 25 mL reaction included 1X Taq buffer, 3.0 mM of magnesium chloride, 0.4 mg/ml of BSA, 0.12 mM of dNTPs, 0.6 mM of each primer, 1 unit Taq DNA polymerase and 1 mL of extracted DNA. PCR conditions were one cycle of 94uC for 3 min, 35 cycles of 94uC for 30 s, 46uC for 30 s and 72uC for 90 s, and a final extension step of 72uC for 7 min. The purification and sequencing were conducted by Macrogen Inc. (Korea, Seoul) and using an AB3500 automated sequencer (Applied Biosystems) in the Laboratório de Biodiversidade Molecular, Federal University of Rio de Janeiro (Brazil).
The electropherograms were edited with Sequencher 4.1 software (Gene Codes Corporation) and sequences were aligned in MEGA 5.0 [13] with ClustalW tool. The number of divergent sites and the intra and inter-specific genetic distances were also calculated with MEGA 5.0 [13]. Sequences were deposited in the GenBank database (www.ncbi.nlm.nih.gov/genbank).

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: 2469618D-EEF2-49F8-95A9-E74C0B83F65E. 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.

Taxonomic Account
Key to genera and species of multitentaculate Cirratulidae from South America -Preserved specimens with dark brown lateral stripes on tentacles and branchiae and dark spots throughout the body .
..Timarete punctata species complex. 7(6) Branchial filaments on mid-body and posterior chaetigers shifted to high on body wall above notopodia; distance between same segment branchial pairs smaller than distance in between each filament and notopodial base; posterior neuropodia with single large, dark spine on posterior segments lacking companion capillaries .
-Branchial filaments shifted to mid-dorsum but gap between same segment branchial pairs always larger than distance in between each filament and notopodial base; posterior neuropodia usually with 2-
Prostomium conical, as long as two anterior chaetiger, with pair of large postero-lateral nuchal organs, as deep depressions ( Fig. 2A). Peristomium elongate, as long as four anterior chaetigers with three similar-sized annulations; posterior two annuli with 3-4 sub-annulations ( Fig. 2A). Branchial filaments first present on posterior end of chaetiger 1, one pair per segment, present in most segments on first one-third of body, few afterwards, absent on last 20-30 chaetigers. Two oblique groups of tentacular filaments arise in two groups on each side of chaetigers 5-6 or 6-7, each with 20-25 tentacles (Fig. 2B).

Methyl Green Staining Pattern
Distinct staining reaction on prostomium, leaving dorsal region unstained, peristomium staining dorsally and laterally. Body with segmental regions with stain forming complete rings around individual segments, inter-segmental grooves not staining (Fig. 3B); green speckled rings more evident on posterior segments.

Habitat
Intertidal depths in sandy substrate.

Remarks
Cirriformia capixabensis, sp. nov. is most similar to C. tentaculata (Montagu, 1808) and C. pygidia (Treadwell, 1936) by the distribution of feeding tentacles above chaetigers 5-6. C. pygidia is unique in the genus by the absence of notopodial spines, present in all other described species (e.g. Table 1). The early segmental origin of the neuropodial spines in C. tentaculata (ch. 25) is the most distinctive feature in comparison with C. capixabensis, sp. nov. (ch. 34-40).
The segmental origin of the dorsal tentacles differs in Cirriformia capixabensis, sp. nov. from that of C. chicoi, sp. nov., with the former having a posterior segmental origin of these tentacles at chaetigers 5-6 or 6-7 while C. chicoi, sp. nov. has tentacles from chaetigers 2-3, 3-4 or 4-5. Both species had MGSP forming complete rings on certain the body segments, on C. chicoi, sp. nov. the bands are limited to the posterior half of individual segments while in C. capixabensis, sp. nov., the entire segment is stained leaving only the inter-segmental grooves unstained. See Table 1 for comparison with other Cirriformia species from the Atlantic Ocean and molecular identity section for a molecular comparison between Cirriformia capixabensis, sp. nov. and C. chicoi, sp. nov.

Etymology
This species is named after the type locality and the term 'capixaba' refers to those who are born in the Espírito Santo state.

Reproduction
One female was collected with oocytes of about 70 mm in diameter. Asexual reproduction or regenerating individuals were not observed.

Molecular identity
COI fragments of 658 bases pair (bp) were obtained from three individuals, one from Espírito Santo, Vitória, Boi Island and two from Espírito Santo, Guarapari. These sequences were deposited in GenBank under the accession numbers, KM192161-KM192163. The three COI sequences have no differences. See Molecular identity in C. chicoi, sp. nov. for inter-specific comparisons between the two Cirriformia species.
Prostomium elongate, as long as four anterior chaetigers, with pair of postero-lateral nuchal organs (Figs 4A, B, 5A). Peristomium with three annulations, as long as six anterior chaetigers; last annulus slightly longer than other two (Figs 4A, B, 5A). Branchial filaments first present on posterior end of third peristominal annulus (Fig. 5B); present in most segments on first one-third of body and absent on last one-third. Two oblique groups of tentacular filaments arise on each side of chaetigers 4-5 (or 3-4 on smaller specimens) with 18-20 (Fig. 4B).

Methyl Green Staining Pattern
Anterior end, including prostomium and peristomium, staining lightly. Mid-body region and posterior end stained with transversal bands on posterior half of segments forming complete rings (Fig. 3A); bands more evident on last third of the body.

Habitat
This species was collected associated with the calcareous alga Halimeda opuntia on the edges of tidal pools at intertidal depths and on soft bottom sediments.

Remarks
This species is unique in that juvenile individuals had bidentate acicular spines, whereas mature individuals had sub-dentate spines. The development of C. moorei (as C. spirabrancha) was studied by [14] and all juveniles were found to have bidentate spines; in adults, these spines were unidentate. This has also been reported in C. tentaculata [15] and may be universal during the early development of multitentaculates.
Cirriformia chicoi, sp. nov. is most similar to Cirriformia grandis (Verrill, 1873) by the peristomial origin of the branchial filaments and segmental origin of spines on notopodia and neuropodia. It differs most noticeably by the segmental origin of the feeding tentacles, on chaetiger 1 in C. grandis and on chaetigers 3-4 or 4-5 in C. chicoi, sp. nov. and by the shape of the spines being heavy and unidentate in C. grandis and slender and knobbed-like in adults of C. chicoi, sp. nov. Table 1 shows morphological characteristics of all Cirriformia species described from the Atlantic ocean for comparison.

Etymology
This new species is named after Dr. Francisco Barros (a.k.a. Chico), a Brazilian Benthic Ecologist who introduced and motivated the author (WFM) to the study of polychaetes. The species Pentacirrus julianae Wesenberg-Lung, 1958 described from the Lesser Antilles was not included as it needs to be revised and the type may be a juvenile individual; distribution of feeding tentacles in two rows above several segments resembles those in Protocirrineris species although curved spines are present. doi:10.1371/journal.pone.0112727.t001 Sexual reproduction occurs for this species as some females were collected bearing oocytes of 70-80 mm in diameter. Asexual reproduction or regenerating individuals were not observed.

Molecular identity
COI fragments of 658 bp and a 16S fragment of 522 bp were obtained from two and one individual, respectively. All individuals were from Rio de Janeiro, Cabo Frio, Japonês Island. The COI and 16S sequences were deposited in GenBank under the accession numbers, KM192164-KM192165 and KM192189, respectively. The two COI sequences differed by 7 bp (p-distance and K2P = 1.1%). The COI inter-specific genetic distance between C. chicoi, sp. nov. and C. capixabensis, sp. nov. was of 22.1% -26.3% (p-distance -K2P).

Distribution
This species is known from northern and southern Brazil (Fig. 1). The type locality is Itapuã Beach in Salvador, Bahia state.

Description
Specimens 9-38 mm long, 1-4 mm wide for 68-192 chaetigers. Body in cross-section, dorsally rounded and ventrally flat; larger specimens (.30 mm) with shallow or deep ventral groove from middle to posterior end. Color in alcohol pale yellow to light brown, ventral part of prostomium, peristomium and first three chaetigers with dark pigmentation; in life, with body, branchiae and feeding tentacles orange to reddish (Fig. 7A); some specimens with darker pigmentation on dorsum and anteriorly on ventrum. Pygidium with small ventral lobe and dorso-terminal anal aperture (Fig. 6C).
Prostomium short, conical with rounded end, twice as wide as long; eyes absent, nuchal organ depression postero-lateral (Fig. 6A). Peristomium also short, as long as two anterior chaetigers with three similar sized annulations; peristomial annuli well-separated in large individuals (Fig. 6A, D). First pair of branchial filaments arise on posterior end of third peristomial annulus; branchiae one pair per segment (but see Remarks) on anterior one-third of body, few on mid-body segments and absent on last one-third. Branchiae shift abruptly to mid-dorsum forming lateral bulge over notopodia (Fig. 6D); segmental origin of branchiae related to size, ranging from chaetigers 10-26 (see   8B). On largest branchial shift, space in between same segment branchial pairs smaller than space between each filament and notopodial base; last branchial filaments still arise from middorsum, not returning to near notopodial base. Two oblique groups of about 10-16 tentacular filaments arise above two or three chaetigers, e.g. 3-4, 4-5 or 5-7 (Fig. 6A).

Methyl Green Staining Pattern
No distinctive staining pattern; entire worm stains light green.

Habitat
This species has been found on calcareous encrustations and soft bottom sediments from intertidal depths to shallow subtidal zones.

Remarks
Syntypes of Timarete caribous were examined by [19] and they fully agree with the material from the USA east coast, Colombia, and northern and southern Brazil. There is some size-related variation on segmental origin of dorsal tentacles, noto-and neuropodial spines and branchial shift to mid-dorsum. Specimens undergoing asexual reproduction may even increase the variability of these characters. This species is easily distinguishable from other Timarete species by the presence of a large, dark and single neuropodial spine on posterior segments and position of branchial filaments on mid-body and posterior segments at a considerable distance from notopodia. The branchial filaments in this species occur as a single pair per segment but some regenerating anterior ends had more than a pair per segment.

Reproduction
Many specimens were found with the anterior and/or posterior end in process or completely regenerated (e.g. Fig. 7B). Therefore, asexual reproduction is likely to occur in this species. A female with oocytes measuring 80-100 mm in diameter has been reported in [19].

Molecular identity
COI fragments of 658 pb were obtained from 12 individuals, three of each locality: São Pedro e São Paulo Archipelago, Fernando de Noronha Archipelago, Rocas Atoll and Abrolhos Archipelago. Whereas a 16S fragment of 526 bp were obtained from four individuals, three from Fernando de Noronha Archipelago and one from São Pedro e São Paulo Archipelago. The COI and 16S sequences were deposited in GenBank under the accession numbers, KM192166-KM192177 and KM192190-KM192193, respectively. The COI sequences showed 22 variable sites and the maximum difference between two sequences were 19 bp (SPSPA 6 AA). The intra-specific genetic distance varied from 0%-2.9% (p-distance) and from 0%-3.0% (K2P). The 16S sequences showed 7 variable sites and the maximum difference between two sequences were 6 bp (SPSPA 6 FNA).

Distribution
The type locality is St. Croix in the Caribbean. This species seems to be widely distributed in the northwestern, central and southwestern Atlantic Ocean. Several specimens from Florida Keys and Colombia, previously identified as T. filigera, actually belong to T. caribous. In Brazil, this species is widely distributed and has been collected in the northern (Pernambuco and Bahia) and southern states (Rio de Janeiro and Santa Catarina) including oceanic Islands (São Pedro e São Paulo Archipelago, Fernando de Noronha and Rocas Atoll). This species is reported as an invasive species that has entered the Mediterranean through ballast water [19].

Description
Holotype 35 mm long, 1.8 mm wide for 165 chaetigers. Paratypes ranging from 9-34.5 long, 0.6-2 mm wide for 94-165 chaetigers. Body in cross-section, dorsally rounded and ventrally flattened with a ventral groove formed by projection of parapodial shoulders. Color in alcohol pale yellow or light grey; some individuals with ventral pigmentation; tentacles and branchiae slightly lighter than body; color in life, yellowish to light orange with branchiae lighter than body and feeding tentacles, some branchiae with whitish areas (Fig. 7C, D). Pygidium with either terminal anus or small ventral lip with dorso-terminal anal aperture.
Prostomium conical, rounded anteriorly, as long as two anterior chaetigers (Figs 7C; 9A, B); eyes absent, nuchal organ depression postero-lateral. Peristomium as long as four anterior chaetigers, with three similar-sized annulations; first peristomial annulus dorsally inflated forming crest (Fig. 9B). First pair of branchial filaments arise on posterior end of third peristomial annulus; branchiae one pair per segment on anterior one-third of body, scarce on mid-body segments and absent on last one-third of body. Branchiae shift gradually to mid-dorsum from chaetigers 8-14 (Fig. 8D), not forming lateral bulge over notopodia; branchiae on posterior segments with distance from notopodial base similar to distance between noto-and neuropodia. Two slightly oblique groups of 5-6tentacles, arise as early as chaetigers 2-3 but larger specimens with tentacles on chaetigers 4-5 (Fig. 9B).

Methyl Green Staining Pattern
Larger specimens with distinct staining reaction (Fig. 3E). Dense green speckles present on individual body segments forming complete rings (Fig. 3F). Branchial filaments also stained with green speckles. Pygidium stained with uniform green.

Habitat
This species is found abundantly in muddy sand in intertidal regions of Ribeira Beach, northern Brazil.

Remarks
Timarete ceciliae, sp. nov. has been collected together with specimens of T. punctata complex and T. caribous. It differs from species of the T. punctata complex by the absence of body pigmentation and from T. caribous by the segmental origin of feeding tentacles, branchial shift, and presence of 2-3 neuropodial spine on posterior segments. T. caribous is unique by the presence in posterior segments of single heavy spines and absence of companion capillaries.
T. ceciliae, sp. nov. is most similar to T. hawaiensis  and to material examined of T. nr. filigera (Delle Chiaje, 1828) from the Mediterranean by the presence of feeding tentacles above two anterior segments, usually chaetigers 3-4. The segmental origin of spines in notopodia and neuropodia is also overlapping but spines in T. ceciliae, sp. nov. appear several segments before than in both T. hawaiensis and T. nr. filigera (see Table 2). The main distinctive feature is the abrupt shift in the position of branchial filaments toward mid-body segments in T. hawaiensis and T. nr. filigera and gradual shift in T. ceciliae, sp. nov. More distinctive morphological features can be found in Table 2.

Etymology
This species is named in honor of Dr. A. Cecília Amaral from the Universidade de Campinas, São Paulo, Brazil, for her great contributions to the taxonomy of polychaetes and benthic ecology in Brazil.

Reproduction
Several individuals Timarete ceciliae, sp. nov. were collected regenerating either the anterior and/or posterior ends. Regenerating ends were always lighter in color than the rest of the body. Individuals with a regenerating anterior end may have acicular hooks starting on the segment of fission in both noto-and neuropodia. These individuals were excluded from the data recorded for Fig. 8. One individual had a fully regenerated prostomium and peristomium, feeding tentacles and first pair of branchiae, and noto-and neuropodial acicular spines from chaetiger 1.

Molecular identity
COI fragments of 658 bp and 16S fragments of 515 bp were obtained from two and three individuals, respectively. All individuals were from Pernambuco, Fernando de Noronha Archipelago, Sueste Beach and Bahia, Salvador, Ribeira Beach. The COI and 16S sequences were deposited in GenBank under the accession numbers, KM192178-KM192179 and KM192194-KM192196, respectively. The two COI sequences have no differences. The 16S sequences showed one variable site, and the genetic distance between sequences varied from 0% to 0.2% (p-distance and K2P). The inter-specific genetic distance between T. ceciliae and T. punctata complex was 22.2%-26.5% (p-distance -K2P) for COI and 19.8%-23.2% (p-distance -K2P) for 16S. See Molecular identity in T. caribous for an inter-specific comparison between the T. ceciliae and T. caribous.

Distribution
Type locality is Ribeira Beach in Salvador, Bahia, northern Brazil. This species was also collected further south in the Espírito Santo state.

Description
Holotype missing pygidium, 110 mm long, 7 mm wide for about 310 chaetigers. Paratype complete with pygidium, 85 mm long, 5 mm wide for about 300 chaetigers. Color in alcohol pale yellow, tentacles and branchiae slightly lighter than body. Pygidium with terminal anus in paratype. In cross-section, body dorsally rounded, ventrally flattened with shallow ventral groove.

Methyl Green Staining Pattern
Prostomium and peristomium stained with dark green (Fig. 3C). Body staining lightly with green speckles. Parapodial regions staining with dark green except for notopodial and neuropodial ridges (Fig. 3D).

Habitat
Intertidal and shallow subtidal depths.

Remarks
This species is herein considered as valid and transferred to the genus Timarete because the branchial filaments shift to middorsum of body although not forming a lateral bulge over notopodia. Audouinia oculata was considered a synonym of T. filigera [20]. Type material of T. filigera is considered to be lost but comparative material from the Mediterranean identified as the latter was examined and shows several morphological differences. Table 2 shows several morphological characters for T. oculata, comb. nov. and its congeners. For instance, the Brazilian species T. oculata differs from T. filigera by the following characteristics: 1) branchiae shift abruptly to mid-dorsum from chaetigers 15-20 forming a bulge over notopodia in T. filigera and gradually from chaetigers 20-25 not forming a bulge over notopodia in T. oculata; 2) tentacular filaments arise on chaetigers 3-4 in T. filigera and 4-5 in T. oculata; 3) notopodial acicular spines start from chaetigers 27-31 in T. filigera and from chaetigers 57-58 in T. oculata; 4) neuropodial acicular spines start from chaetigers 15-21 in T. filigera and 38-40 in T. oculata; and. 5) the pygidium has a dorsal anal aperture with a small ventral lip in T. filigera and terminal anus in T. oculata.
The holotype of Cirratulus flavescens Grube 1872 described from Brazil was considered a homonym by [21] probably because the name was preoccupied by C. flavescens Johnston, 1825. This holotype was examined and it belongs to Timarete oculata comb. nov. The specimen is complete and well-preserved measuring 82 mm long, 3 mm wide for about 300 chaetigers. The prostomium is short and rounded; the peristomium has three annulations, with the third annulus twice as long as the first annuli. Branchial filaments arise from the posterior end of the third peristomial annulus and are absent on the last 50 chaetigers. Branchiae gradually shift to the mid dorsum from about chaetiger 50. Neuropodial hooks occur from chaetiger 48 and notopodial hooks from chaetiger 75.

Reproduction
Holotype and paratype were females and coelom was filled with eggs.   Ventral lip and dorsal anal aperture This study; [24] The species Timarete anchylochaeta (Schmarda, 1861) from New Zealand and Timarete japonica Zachs, 1933 from Japan were not included by the lack of detailed morphological information in the original and subsequent descriptions. doi:10.1371/journal.pone.0112727.t002 Description Specimens 1.5-21 mm long, 0.2-2 mm wide for 30-156 chaetigers. Color of living specimens dark yellow with black pigment spots throughout body and branchial filaments; dorsal tentacles with dark rings. Preserved specimens with dark grey body; pigment retained on branchiae and dorsal tentacles. Body in cross section, dorsally rounded and ventrally flat without ventral groove. Pygidium with ventral lip and dorsal anal aperture.
Prostomium short, rounded anteriorly, as long as one or two anterior chaetigers; eyes absent, nuchal organ depression posterolateral. Peristomium as long as 2-3 anterior chaetigers, with three similar sized annulations; second and third peristomial annuli with dorsal and ventral sub-annulations. First pair of branchial filaments arise on posterior end of third peristomial annulus; branchiae with one pair per segment on anterior one-third of body, few on mid-body segments and absent on last 10-20 segments. Branchiae shift gradually towards mid-dorsum but not reaching middle region, from chaetigers 7-18, forming lateral bulge over notopodia, bulge sometimes not observed; branchiae on very posterior end arise from a small distance above notopodial base. Two oblique groups of about 10-14 tentacular filaments arise on chaetigers 3-4 or 4-5.

Methyl Green Staining Pattern
No distinctive staining pattern; entire worm stained with light green.

Habitat
This species was collected associated with the calcareous alga Halimeda opuntia on the edges of tidal pools and on sandy and muddy sediments.

Remarks
These specimens of Timarete punctata are similar to the redescription of the type material by [24] but some variation on body morphology and coloration pattern was found. Juvenile specimens with elongated black spots over the body and mature specimens with rounded black spots on a dark brownish body were described in [24]. This author also observed densely pigmented branchial filaments and tentacular filaments with black lateral stripes. The material collected in Brazil is believed to belong to at least two different species based on pigmentation of live and preserved specimens and on body morphology. However, there is a need for molecular evidence to help distinguish the suspected cryptic species within the T. punctata species complex. Since it is beyond the scope of this study, we designate the species collected along the Brazilian coast as T. punctata species complex until further integrated studies are performed.

Reproduction
Some specimens were found with anterior and/or posterior end in process or completely regenerated. Therefore, asexual reproduction is likely to occur in this species as also reported by [18]. Juvenile individuals (30-40 chaetigers) were also collected indicating the occurrence of sexual reproduction. One specimen from the Mediterranean was collected with oocytes in the coelom having a mean diameter of 65.5 mm [24].

Molecular identity
COI fragments of 633 bp and 16S fragments of 533 bp were obtained from 12 individuals, three of each locality: São Pedro e São Paulo Archipelago, Rocas Atoll and Bahia, Salvador, Ribeira Beach. The COI and 16S sequences were deposited in GenBank under accession numbers, KM192180-KM192188 and KM192197-KM192205, respectively. All COI sequences are identical. The 16S sequences showed one variable site, and the genetic distance between sequences varied from 0% to 0.2% (pdistance and K2P). See Molecular identity in T. caribous and T. ceciliae for an inter-specific comparison between T. punctata complex 6 T. caribous and T. punctata complex 6 T. ceciliae, respectively.

Distribution
Timarete punctata is a complex of species and it has been recorded throughout the Atlantic (western and eastern), Indian (western) and Pacific (western) oceans, e.g. [18,24,25,26]. Preliminary molecular analysis has shown that the Caribbean and South Atlantic material are composed of two distinct species and additional research is planned to elucidate the identity of both species.