Ampharete sp.

Ampharete sp. : Parapar et al. (2009).

Diagnosis: A medium-sized species of up to 21 mm in length and 2.5 mm in width. Branchiae arranged in two well-separated groups. Paleae thin and slender with filiform tips, 9–12 on each side. Twelve thoracic uncinigers and 12–13 abdominal uncinigers. Pygidium with two lateral cirri and several small rounded papillae. Single pair of prostomial eyes; no pygidial eyes.

Material examined

Type material: NW Iberian Peninsula, Atlantic coast of Spain. Holotype: Ría de Ferrol (Galicia, NW Spain), Museo Nacional de Ciencias Naturales, MNCN 16.01 About MNCN /17841 (site 62). Paratypes: Ría de Ferrol : Museo Nacional de Ciencias Naturales , MNCN 16.01 About MNCN /17842 (site 25, 167 specimens) ; MNCN 16.01 About MNCN /17843 (site 38, 148 specs) ; MNCN 16.01 About MNCN /17844 (site 42, 99 specs) ; MNCN 16.01 About MNCN /17845 (site 45, 229 specs) ; MNCN 16.01 About MNCN /17846 (site 55, 2 specs) ; MNCN 16.01 About MNCN /17847 (site 57, 239 specs) ; MNCN 16.01 About MNCN /17848 (site 59, 3 specs) ; MNCN 16.01 About MNCN /17853 (site 59, 2 specs, in SEM stub) ; MNCN 16.01 About MNCN /17849 (site 62, 44 specs) ; MNCN 16.01 About MNCN /17854 (site 62, 3 specs, in SEM stub) ; MNCN 16.01 About MNCN /17850 (site 63, 635 specs) ; MNCN 16.01 About MNCN /17851 (site 71, 21 specs) ; MNCN 16.01 About MNCN /17852 (site MS, 1 spec. scanned for micro-CT) ; MNCN 16.01 About MNCN /17855– 17861 (site 71, 1 spec. sectioned for histology) ; ZMBN 115540–115542 View Materials (DNA vouchers) .

Non-type material: Ría de Ferrol (Galicia): site 6, 11 specs; site 11, 1 spec.; site 12, 1 spec.; site 16, 2 specs; site 18, 49 specs; site 20, 10 specs; site 21, 82 specs; site 23, 1 spec.; site 24, 2 specs; site 26, 5 specs; site 27, 63 specs; site 33, 2 specs; site 36, 123 specs; site 37, 20 specs; site 41, 50 specs; site 44, 491 specs; site 47, 3 specs; site 48, 90 specs; site 49, 95 specs; site 50, 8 specs; site 51, 3 specs; site 52, 1 spec.; site 53, 8 specs; site 54, 30 specs; site 58, 25 specs; site 66, 1 spec.; site 68, 108 specs; site 69, 3 specs; site 72, 34 specs; site Z4 R1 , 1 spec.; site AN, 1 spec. Ría de Aldán (Galicia): site 27, 1998: May, 23 specs; June, 2 specs; July, 69 specs; August, 72 specs; September, 48 specs; October, 85 specs; November, 7 specs; December, 49 specs; 1999: January, 75 specs; February, 52 specs; March, 14 specs; April, 14 specs; May, 36 specsite SITE 31 , 1998 : May, 25 specs; June, 65 specs; July, 148 specs; August, 66 specs; September, 27 specs; October, 79 specs; November, 120 specs; December, 33 specs; 1999: January, 75 specs; February, 149 specs; March, 92 specs; April, 42 specs; May, 295 specs. Ensenada de Baiona (Galicia): site 2, 2 specs; site 4, 1 spec.; site 10, 1 spec.; site 17, 1 spec.; site 18, 2 specs; site 19, 1 spec.; site 20, 1 spec. Morocco: site GR37, 1 spec. ( ZMBN 116931 View Materials ) ; site GR44, 17 specs ( ZMBN 116932 View Materials ) ; site GR45, 2 specs ( ZMBN 116933 View Materials ) and 1 spec. (DNA voucher, ZMBN 98169 View Materials ) ; site GR50, 4 specs ( ZMBN 116934 View Materials ) and 1 spec. (DNA voucher, ZMBN 98164 View Materials ) ; site GR56, 3 specs ( ZMBN 116935 View Materials ) and 1 spec. (DNA voucher, ZMBN 98157 View Materials ) .

Additional studied material of other species: Off Iceland. Ampharete finmarchica (M. Sars, 1865) . MNCN 16.01/15486 (BIOICE sample 2377, 1 spec.), MNCN 16.01/15487 (BIOICE sample 3252, 1 spec.), MNCN 16.01/15488 (BIOICE sample 2660, 3 specs), MNCN 16.01/15489 (BIOICE sample 2070, 4 specs). Specimens reported by Parapar et al. (2012). Ampharete lindstroemi Malmgren in Hessle, 1917. MNCN 16.01/15329 (BIOICE sample 2622, 2 specs), MNCN 16.01/15330 (BIOICE sample 3028, 1 spec.). Specimens reported by Parapar et al. (2012). West Africa. Ampharete debrouweri Jeldes & Lefevere, 1959 . Holotype, PY-987, I.G. 20403, BS-551/ 12.09.1955. Norway. Specimens of A. falcata , A. finmarchica , A. lindstroemi , A. octocirrata and A. undecima used for DNA barcoding ( Table 4).

External morphology (based on holotype): Complete specimen of 18 mm length and 1.5 mm width. Thorax thicker than abdomen and of same length ( Figs 2A, 4A). Prostomium with median lobe delimited by deep lateral grooves; prostomial glandular ridges absent ( Figs 2B, 4A, B, 6A, B, 10A). Two small black, circular eyespots located posteriorly on median prostomial lobe, next to lateral grooves ( Fig. 11A). Nuchal organs not observed. Buccal tentacles with two rows of short papillae on one side ( Fig. 6C–F). Peristomium and segment I only visible laterally and ventrally; peristomium forming well-developed lower lip ( Fig. 6A). Four pairs of branchiae arranged in two groups with small median gap of about one to two times branchial width ( Fig. 2B, C); branchiophores fused at base; branchiae thick at base and gradually tapering towards distal end, less than 1/6 of body length, reaching about TC6; with parallel ciliated rings from base to distal end ( Figs 2A–C, 4B, C, 6A, B, D, 7A, 11A, C). Anterior three pairs of branchiae arranged in transverse row on segment II/III, fourth pair slightly posterior to anterior row, between second outermost and innermost branchiae. Branchiae of segment II in outermost position of anterior row, branchiae of segment III in mid-position of anterior row, branchiae of segment IV in innermost position of anterior row, branchiae of segment V in posterior position. Single pair of nephridial papillae located dorsally on segment IV ( Fig. 2C). Dorsal part of thorax, behind branchial region, covered by patches of ciliated rounded tufts (only seen with SEM). Segment II with 12–15 long, flattened chaetae (paleae), with curved filiform tips, arranged in semicircle ( Figs 6A, 7E, F, 10A). Thorax and abdomen of similar length; thorax slightly wider than abdomen; abdomen tapering posteriorly. Continuous ventral shields present to thoracic unciniger ten ( Figs 5B, 11F). Fourteen thoracic segments with notopodia and capillary chaetae; last 12 also with neuropodial tori bearing single row of uncini. Notopodia as simple lobes, up to three times longer than wide; first notopodium somewhat reduced ( Figs 2A, 5A, B, 6A, B, 8A, B). Notochaetae as simple spinulose capillaries ( Fig. 8C), tapering into slender tips; notochaetae arranged in rows, capillaries from anterior row generally thinner and shorter than in posterior row. Anterior neuropodia oval, about three times higher than wide ( Figs 2A, 3A); neuropodia gradually decreasing in size, becoming more rounded in posterior part of thorax ( Fig. 2A). Thoracic uncini with about ten teeth in two vertical rows above rostrum and basal prow ( Figs 8D, 10B). Twelve abdominal uncinigers ( Fig. 2A). Anterior two abdominal segments with neuropodia of thoracic type ( Figs 8E, 9A, B); remaining abdominal uncinigers with enlarged neuropodial pinnules, with short neuropodial cirrus ( Figs 3B–E, 8E). Abdominal uncini with about five teeth in two vertical rows above rostrum and basal prow ( Figs 9C, D, 10C, F). Ciliated tufts between parapodial rami in thorax on ventral bases of notopodia, and above neuropodia in abdomen (on parapodial ridge) ( Figs 8E, 10E). Pygidium as short cylindrical tube, about as long as wide, with pair of lateral cirri of same length as width of last chaetiger and with about eight pygidial papillae ( Figs 3F–H, 9E, F, 10D, E). Colour in life bright orange, epiderm transparent; thick dorsal blood vessel distinct in anterior dorsal thorax as deep red tube ( Fig. 11A–C). Specimens in alcohol pale orange to yellow, with opaque epidermis ( Fig. 4). Tube more or less quadrangular in cross section, incrusted with mud and relatively large pieces of shell fragments ( Fig. 11E). Head and ventral shields deeply dyed in methyl blue ( Fig. 11F).

Variability: Complete specimens measure 11–21 mm in length and 0.5–2.5 mm in width. Several specimens from Galicia bear 13 abdominal chaetigers instead of 12. The pygidium showed variability regarding the number of papillae and length of lateral cirri; however, the actual variability of these characters is difficult to ascertain because the pygidium is often contracted in fixed specimens, sometimes inside the posterior end. Branchiae seem longer in live compared to fixed specimens (see Fig. 11A–C vs. Figs 2A, B, 4A–D, 6A, B), probably due to their intrinsic flexibility, and contraction caused by fixation. Oocytes were seen filling the coelomic cavity in live specimens ( Fig. 11B).

Anatomy ( Figs 5, 12 – 15): The micro-CT 3D reconstructions of internal longitudinal and transversal sections show the shape and position of musculature and organs in high quality, primarily those of muscular, digestive, circulatory, excretory and reproductive systems. The integument comprises the cuticle, epidermis and thin longitudinal musculature bands and is shown to be thicker ventrally (in thorax) due to the glandular epidermis ( Fig. 12). The digestive system fills most of the coelomic cavity and has a wide anterior buccal cavity (pharynx or fore digestive) filled with retracted buccal tentacles in this specimen ( Figs 5C, D, 12A–C), a short oesophagus connecting with a large stomach (median gut) supported by a muscular ventral mesentery ( Fig. 12D, E), and a long, narrow and internally looped intestine (hind gut) ( Figs 5C, D, 12F). The buccal cavity contains a ventral pharyngeal organ ( Figs 5C, 13B) that is also discernible with SEM ( Fig. 6D, E). The stomach shows an anterior pair of lobes surrounding the oesophagus ( Figs 12C, D, 13A) followed by a continuous tube with irregularly distributed constrictions that may be related to the retraction of the buccal cavity ( Fig. 5C, D). Transversal views do not show the circular musculature of the body wall; however, many thin bundles of longitudinal muscles can be seen dorsally and ventrally ( Figs 13A, 14B, C). Thin bundles of oblique parapodial musculature connect the basal part of thoracic notopodia with the inner ventral body surface ( Fig. 12E, F). The circulatory system is represented by a well-developed dorsal blood vessel, which can be best seen anteriorly ( Figs 5C, 12B–D, 14A, B), and an expanded blood sinus completely covering the stomach ( Figs 12D, E, 13A, B). The only conspicuous part of the central nervous system is the nerve cord located ventrally, close to the integument ( Fig. 12D). No brain or circumpharyngeal connectives were observed. Tissue masses located ventrally to the buccal cavity and stomach in the median thoracic region may be related to segmental organs ( Figs 5C, 12B–E); their shape probably corresponds to different biological roles. Histological body sections of paratypes 16.01/17855 to 16.01/17860 taken from the anterior thoracic region ( Fig. 15A) provide complementary information to that of micro-CT. Images show the presence of several anatomical structures inside a coelomic cavity partially filled with oocytes in different stages of maturation ( Fig. 15B). Those structures are the stomach ( Fig. 15B), the lateral branches of the segmental organs-nephridial ducts ( Fig. 15C), the ventral nerve cord ganglion ( Fig. 15D) and the stomach blood sinus ( Fig. 15E).

Molecular results ( Fig. 16, Table 2): All morpho-species were recovered as monophyletic ( Fig. 16) with low genetic intraspecific K2P variation (<0.5% in COI), except for A. acutifrons where the single specimen from Alaska differed from the four specimens from California by 7.1% ( Table 2). The mean interspecific K2P divergence between species was 17.4%, ranging from 3.3% between A. manriquei and A. octocirrata to 21.6% between A. santillani sp. nov. and A. falcata ( Table 2). Ampharete santillani sp. nov. was recovered as sister species to A. lindstroemi with high bootstrap support, and the intraspecific distance between these two species was 15.1% ( Table 2).

Epibiosis ( Figs 6A, 7B–D, 8A, B, E, F, 10E, F, 17): A high infestation by ciliated protozoans on the external body surface was observed in specimens from both the Ría de Ferrol ( Figs 6–8, 17) and Morocco ( Fig. 10). Although they have been found in many body parts, such as attached to the abdominal uncini ( Fig. 10E, F), their abundance seems to be higher in ciliated body parts, such as the branchial surface ( Fig. 7B–D), the dorso-lateral area behind them ( Figs 6A, 8A, B) and the ciliated buttons over the abdominal neuropodia ( Fig. 8E, F).

Type locality: Ría de Ferrol (Galicia, NW Spain), 43°28′46″N, 08°11′45″W, sandy mud, 1.5 m depth, organic content in sediment = 3.22 GoogleMaps %.

Ecology ( Figs 18 – 19): The new species was found in the Ría de Ferrol, Ría de Aldán and Ensenada de Baiona (NW Spain) mostly in fine sand and muddy sediments (sandy mud to mud) at inner areas of these rias at depths of between 1 and 20 m ( Table 1); some specimens were occasionally found in gravel and coarse sand at the outer half of the Ría de Ferrol ( Fig. 18). Specimens from Morocco were found in slightly deeper waters (36–106 m) than in NW Spain; however, no samples from shallower localities (<30 m) were available from Morocco. Organic content of the sediment ranged from 0.3 to 15.1% and 0.3 to 8.5% in Ferrol and Baiona, respectively; in Aldán, the values in site 27 (muddy sand), ranged between 0.9 and 4.8%, while in site 31 (mud), they were much higher: 9.9–15.1%. There were, however, no significant correlations between organic matter and monthly abundance at any site (Spearman correlation coefficient, P> 0.05, d.f. = 11). Temporal variation in the Ría de Aldán showed that the new species may reach densities of up to 500–1000 ind./m 2 in site 31 and up to 300 ind./m 2 in site 27 ( Fig. 19). In site 27, peaks of abundance were detected in summer/autumn and January, while in site 31, peaks (i.e. July, November, February, May 1999) were followed by conspicuous decreases in the following months.

Distribution: NW Iberian Peninsula, off southern coast of Morocco ( Fig. 1). Previous reports of A. acutifrons / A. grubei in the Iberian Peninsula (e.g. Cantabrian coast,

Portuguese coast) may represent A. santillani sp. nov. (see Discussion).

Etymology: The species is named after the late Enrique Santillán (Quique), biologist, teacher at the Marista school of Cristo Rey (A Coruña, Spain) and friend of first author. Quique was an avid collector of marine sands around the world, and his kindness and love for nature has been an inspiration to many generations of young students.

IDENTIFICATION OF NE ATLANTIC SPECIES OF THE GENUS AMPHARETE

A synopsis of diagnostic characters for all 13 valid species of Ampharete occurring in the NE Atlantic (including A. santillani sp. nov.) is given in Table 3. Traditionally, species of Ampharete have been identified based on the number of abdominal uncinigers, arrangement of the branchiae, shape of anal papillae, the shape of neuropodial dorsal cirri and their distribution along the body and the shape of paleae ( Holthe, 1986; Imajima et al., 2012; Parapar et al., 2012). However, accepting Sabellides and Asabellides as junior synonyms of Ampharete (see Introduction), the presence/absence of paleae and number of thoracic uncinigers are also relevant characters to distinguish between species of Ampharete occurring in the NE Atlantic. Furthermore, we regard the presence and number (pairs) of prostomial and pygidial eyes as important characters to distinguish between certain species.

Ampharete minuta View in CoL , originally described from the Madeira Islands and not reported after its original description, was not included in the table. The type material could not be located, and the original description by Langerhans (1881) lacks detailed information about several taxonomical important characters (see Discussion). Ampharete grubei Malmgren, 1866 View in CoL , originally described from Greenland and N Europe, has long been considered as a synonym of A. acutifrons View in CoL . However, Jirkov & Leontovich (2013) questioned this and suggested that this taxon should be reinstated. Pending a revision of the type material, we chose not to include it in the synoptic table presented here.