Diopatra micrura

Diopatra micrura, sp nov. Figs. 1–8; Tables 1 –3 Type material. Holotype: MNHN MB 29 -000166, Sta. RA 4 (Nov-09) (incomplete specimen, 51 mm long (61 chaetigers), 3.3 mm wide). Paratypes: MNHN MB 29 -000167, Sta NS (1); MNHN MB 29 -000171, Sta.RA 1 (1); MNHN MB 29 - 0 0 0 168, Sta TS 14 B (1); MNHN MB 29 -000170, Sta TS 23 C (1); MNHN MB 29 -000169, Sta TS 24 (1); MNCN 16.01 / 11627, Sta. TS 1 A (1); MNCN 16.01 / 11628, Sta.TS 3 A (1); MNCN 16.01 / 11629, Sta. TS 4 A (1); MNCN 16.01 / 11630, Sta. TS 22 (1); MNCN 16.01 / 11631, Sta. TS 25 (1); AM W 36251, Sta. NS (1); AM W 36252 Sta. TS 13 (1); AM W 36253 Sta. TS 23 (2); AM W 36254, Sta. TS 23 C (1); AM W 36255, Sta. RA 2 (1); DBUA- 0 1140.01, Sta. TS 1 B (1); DBUA-01141.01, Sta. TS 12 (1); DBUA-01142.01, Sta. TS 12 A (1); DBUA- 0 1143.01, Sta. TS 14 C (1.); DBUA-01144.01, Sta TS 23 A (1). Etymology. The striped antennae of the new species evoke the pattern of the coral snakes Micrurus spp., hence the name Diopatra micrura, sp. nov. Morphological description. Length of complete preserved specimens from 1.7 to 7.8 cm, number of chaetigers from 70 to 97; width of 10 th chaetiger from 0.6 to 4.5 mm without parapodia. Some incomplete specimens regenerating anterior end of body (paratype AM W 36252); one specimen posterior end. Overall colour of living specimens greenish dorsally, cream ventrally. Antennostyles and palpostyles with very characteristic transverse brown bands, 4–8 on antennae and 2–4 on palps (Figs 2 A–C, 3 A). Frontal lips whitish with brown pigment at base and ceratophores with brown rings (Fig 2 B). Prostomium with brown pigment; area of nuchal grooves paler (Figs 2 B, C). Peristomium with brown pigment (Fig. 2 C, 3 A), peristomial cirri cream. Additionally, anterior 10–15 chaetigers with small iridescent white spots (Fig. 2 C) and following chaetigers with iridescent transverse white line (Fig. 2 A). Laterally, from chaetigers 1–4 to 13–23 two brown patches, one on each side (Figs 2 C, 3 A). Branchiae green, parapodia cream (Fig. 2 D); dorsal cirri with iridescent white spots. In preserved individuals, the body is cream with two brown patches laterally on each segment up to chaetigers 13–23 (Fig. 3 A). Lack of coloration in middle of each chaetiger forming “white” line along body (Fig. 3 A). Brown pigmentation of antennae, palps, ceratophores, prostomium, frontal lips and peristomium noticed in living specimens still present. Prostomium anteriorly rounded with subulate frontal lips (Figs 4 A, B). Ceratophores of antennae and palps with proximal rings and longer distal ring, holotype with 14 proximal rings, other specimens with 12–15 rings (Figs 2 A–C, 3 A, 4 A). Antennostyles relatively long, tapering to distal end, ending in fine point; in holotype laterals reaching chaetiger 9, median reaching chaetiger 5, in other specimens 6–13 and 4–10 respectively; palpostyles shorter, reaching chaetiger 2 in holotype, 2–4 in other specimens. Length of antennae quite variable, apparently unrelated to size of specimens (based on width of 10 th chaetiger) (Fig. 5 A). Sensory buds present on antennostyles and palpostyles forming 12–14 irregular longitudinal rows (Fig. 4 C). Sensory buds slightly raised, with pores forming circles (Fig. 4 D). In addition, randomly distributed sensory buds on ceratophores, frontal lips, upper lips, prostomium, peristomial cirrus, peristomium and branchiae (Figs 4 G, H). Nuchal grooves crescentic (Fig. 4 E). Peristomium as long as first chaetiger, bearing pair of peristomial cirri, about twice as long as peristomium (Fig. 3 A). First four modified parapodia (chaetiger 1 to 4) projecting laterally and slightly anteriorly, slightly longer than following non-modified, laterally projecting parapodia (Figs 4 B, F). Prechaetal lobes rounded, present up to chaetigers 7–11, postchaetal lobes subulate (Fig. 4 F), becoming gradually smaller towards posterior region but still distinct till end of body. Ventral lobes present on chaetiger 5 to 14 – 20, subulate to ovate (Fig. 2 D); most distinct on chaetigers 6–15, then shifting more dorsally, forming new prechaetal lip by chaetiger 20–25. Dorsal cirri subulate, becoming more slender posteriorly; ventral cirri cirriform on first 4 chaetigers. Spiralled branchiae from chaetiger 4 in holotype, chaetigers 4 or 5 in paratypes, best developed from chaetigers 6 to 9 with 8–14 whorls, reaching to prostomium when anteriorly extended (Figs 2 A, C); decreasing gradually towards posterior end, absent from chaetiger 45 in holotype, chaetigers 32–55 in other specimens, depending on size of specimens (Fig. 5 B). Branchial filaments fine and short, only slightly longer than width of branchial stem (Figs 2 D, 3 D). Modified parapodia with 1–2 slender upper limbate chaetae and 5–6 bidentate pseudocompound hooks (Fig. 3 E). Hooks with moderately long pointed hoods (Figs 3 H, 6 A) and two rows of small spines along their shafts (Figs 3 H, 6 B). Remaining parapodia with limbate and pectinate chaetae (Figs 3 D, 6 C, D). Pectinate chaetae flat, with 5–10 long teeth, ending in slender tips (Figs 3 F, 6 E); limbate chaetae with narrow serrated wings, overall spiny (Figs 3 G, 6 F). Starting from chaetiger 11 in holotype, chaetigers 8–13 in other specimens, lower limbate chaetae replaced by 2 thick bidentate subacicular hooks with translucent guards (Fig. 3 I). Slope of the regression line of start of subacicular hooks very close to nil, indicating a non-significant relationship to size of specimens (Fig. 5 B). Pygidium with two pairs of anal cirri; dorsal pair about as long as the last six chaetigers, ventral pair about as long as the two last chaetigers. Mandibles (Fig. 3 C) weakly sclerotised, with slender shafts and strongly calcified cutting plates. Maxillae moderately sclerotised (Fig. 3 B). Maxillary formula (based on 9 paratypes): Mx I= 1 + 1; MxII = 8–10 + 8–11; Mx III = 8–11 + 0; Mx IV = 5–8 + 7–11; Mx V = 1 + 1. Tube characteristic of genus, cylindrical with soft inner secreted layer and outer layer of debris, fragments of sea grass, algae and shells. Width of 10 th chaetiger without parapodia (mm) 0.6–4.5 1.90 0.78 77 Remarks. The intraspecific variability of the major morphological characters of Diopatra micrura is summarised in Table 2 and the comparison with other European Diopatra species in Table 3. The multivariate analysis of the morphological data is shown in Figure 7. The groups of individuals belonging to the various species form distinct clusters (Fig. 7, upper graph), represented by well isolated clouds of points in the ordination diagram (Fig. 7, lower graph). The PCA axis 1 and 2 comprehend 91.3 % of the total variance. Diopatra neapolitana opposes D. marocensis on the ordination axis 1, with D. micrura occupying a transition position, on the positive pole of axis 2. Most of the Diopatra specimens from France form a distinct cluster, isolated in the negative pole of axis 2 but closer to D. marocensis. This cluster includes five specimens from Arcachon Bay and a single specimen from Marennes Oléron (Fig. 7). Nevertheless, two specimens from the Arcachon Bay are plotted together with the cluster of Diopatra neapolitana, indicating the coexistence of at the least two species in this Bay. The morphological descriptors most strongly correlated with PCA axis 1 were the number of branchiae whorls (r = - 0.92), the number of rings in the ceratophores (r = - 0.90) and the presence-absence of ventral lobe in the parapodia 5–20 (r = - 0.85). The chaetiger where the subacicular hooks start (r = - 0.72), the width of the 10 th chaetiger (r = - 0.70) and the number of teeth in the pectinate chaetae (r = - 0.53), were the variables strongly correlated with PCA axis 2, the latter especially related to the Diopatra sp. individuals from Arcachon Bay (Fig. 7). Distribution and habitat. Diopatra micrura occurs along the western and southern Portuguese coast. Specimens were collected in Ria de Aveiro, near the mouth, intertidally and on the adjacent shelf area (A), on the shelf off Nazaré (B), in Guia, off the Tagus Estuary (C), and near the Guadiana river mouth (D) (Fig. 1). The species seems to have a preference for fine sand and shallow waters. In the shelf area off the Tagus estuary, it was found in 22 of the 30 sites comprising the annual monitoring program of this area, carried out since March 1994. Diopatra micrura has been found in every annual sampling campaign, in sites ranging from 40 to 60 metres depth, on fine and very fine sand, with fines content up to 25 % of total sediment. In the Aveiro shelf, D. micrura was found in 8 of 22 sites sampled in 2002, always close to 15 metres depth and in fine and very fine sand with less than 5 % fines content. In the Nazaré shelf, the species was found at 37 metres depth, on fine sand with 7 % fines. On the southern coast, it was found in fine and very fine sand with less than 5 % fines, ranging from 4 to 10 metres depth. Finally, in Ria de Aveiro the species was found in the intertidal region, together with D. marocensis and D. neapolitana, in very fine sand with close to 25 % fines content. Genetic analysis. A 702 -bp COI fragment and a 525 -bp 16 S fragment were successfully obtained from 14 individuals of D. micrura. COI and 16 S nucleotide sequences from D. micrura sp. nov., were deposited at EMBL database, under the accession numbers: 16 S – GQ 456163 and COI – GQ 456161 and GQ 456162. For the 16 S gene, all individuals displayed identical nucleotide sequence but in the case of the COI gene, one individual from Ria de Aveiro presented a base alteration, at position 276, where a nucleotide adenine was replaced by a thymine (ATA to TTA), corresponding to an amino acid alteration (methionine to leucine). All specimens sampled on the shelf off the Tagus Estuary shared the same nucleotide sequence. The percentage of nucleotides divergence of the 16 S and COI genes between D. micrura and D. marocensis was 15 % and 17 %, respectively (nucleotide substitution). For D. micrura and D. neapolitana, the divergence was 16 % for COI and 12 % for 16 S. For COI, deduced amino acid sequence comparison between the species revealed that D. micrura differs from D. marocensis in six amino acids and from D. neapolitana in two amino acids, for one haplotype, and in three for the other, revealing 2.59 % and 1.08 % of divergence, respectively. The majority of the differences in nucleotides between D. micrura and those two species occurred on the third position of the codon and therefore corresponded to silent alterations. Comparing COI and 16 S genes of Diopatra sp. from Arcachon Bay with D. neapolitana, D. marocensis and D. micrura, the percentage of nucleotides divergence varied between 17 % and 19 % in the case of the COI and between 16 % and 19 %, for 16 S gene (nucleotide substitution). The phylogenetic analysis from both genes (Fig. 8) separates the Diopatra species into four clades.Published as part of Pires, Adília, Paxton, Hannelore, Quintino, Victor & -, Ana Maria Rodrigues, 2010, Diopatra (Annelida: Onuphidae) diversity in European waters with the description of Diopatra micrura, new species, pp. 17-33 in Zootaxa 2395 on pages 22-31, DOI: 10.5281/zenodo.19393