Leptostracans ( Crustacea : Phyllocarida ) from the Ría de Ferrol ( Galicia , NW Iberian Peninsula ) , with description of a new species of Nebalia Leach , 1814

1 Estación de Bioloxía Mariña da Graña, Universidade de Santiago de Compostela, Casa do Hórreo, Rúa da Ribeira 1, E-15590, A Graña, Ferrol, Spain. E-mail: juan.moreira@usc.es 2 Departamento de Zooloxía e Antropoloxía Física, Universidade de Santiago de Compostela, Campus Sur, E-15782, Santiago de Compostela, Spain. 3 Instituto de Acuicultura, Universidade de Santiago de Compostela, Campus Sur, E-15782, Santiago de Compostela, Spain.


INTRODUCTION
Leptostracans (Crustacea, Phyllocarida) are characterised by the presence of a hinged rostrum, a carapace that covers the thoracic segments, eight pairs of phyllopodus thoracic appendages and seven abdominal segments (Haney and Martin, 2005).The order Leptostraca comprises about 42 known extant species belonging to 10 valid genera (Haney and Martin, 2004).The species concept for this order was revised by Dahl (1985), who proposes new diagnostic characters useful for the identification of species and for revising and describing several taxa (Dahl, 1985(Dahl, , 1990)).Consequently, the examination of specimens from collections worldwide by other specialists has resulted in the description of a number of new species of Leptostraca (e.g.Kazmi and Tirmizi, 1989;Escobar-Briones and Villalobos-Hiriart, 1995;Martin et al., 1996;Vetter, 1996d;Walker-Smith, 1998;Olesen, 1999;Haney et al., 2001;Moreira et al., 2003a;Haney and Martin, 2004).Examination of further collections will undoubtedly result in the description of new taxa, thus revealing a greater diversity within the group than was previously thought (Haney and Martin, 2005).
Leptostracans are found in a number of marine habitats, such as hard substrata with algae, seagrasses and sediments (Dahl, 1985), and from the intertidal zone to the hydrothermal vents (Haney and Martin, 2005).Some species achieve large densities in some substrata (Martin et al., 1996;Vetter, 1996c) and in conditions of organic enrichment (MacLeod et al., 2007).Recent work suggests that some leptostracans might be found in significant numbers solely in particular environments.For instance, Vetter (1996c) reported two species of Nebalia from Southern California coexisting in the same area but occurring in different sediments; experimental manipulations also showed that those species search for their own preferred sediment, avoiding that of the other (Vetter, 1996c).Nevertheless, our knowledge on life-history and ecology for most of the known species is scarce at best (but see Rainer and Unsworth, 1991;Modlin, 1996;Vetter, 1996aVetter, , 1996bVetter, , 1996c;;Lee and Morton, 2005) and more data are needed for a better understanding of the order.
An examination of samples from subtidal sediments collected in the Ría de Ferrol (Galicia, NW Spain) during several sampling programmes done between 2006 and 2007 yielded specimens of leptostracans belonging to six species.This is, so far, the largest number of species reported from a single area.Some of these specimens belong to an undescribed species of Nebalia Leach, 1814, which is described herein as N. reboredae n. sp.All the species collected in the Ría de Ferrol are reported in this paper, including the number of specimens, sexual condition and type of sediment from which they were collected.A key to all known neritic species of the Iberian Peninsula is provided as well.

MATERIAL AND METHODS
The Ría de Ferrol is located in NW Galicia (Spain), between 43º27' and 43º30'N and between 08º09' and 08º21'W.It is a fully marine environment (salinity: 32-25).It is 15 km long and comprises an area of 25 km 2 , with maximum depths of about 35-40 m.Sampling covered most of the subtidal sediments of the ria (from gravel to mud), except those located in the innermost area.Samples were taken between 2006 and 2007 by means of a naturalist's dredge and two van Veen grabs with a different sampling surface (0.1 m 2 and 0.056 m 2 ).Specimens were fixed in 5% formaldehyde, rinsed with fresh water and then preserved in 70% ethanol.
Leptostracans were found at 11 subtidal stations at depths of between 4 and 41 m (Table 1, 2).For each sampling site, a sediment sample was taken to determine granulometry, calcium carbonate (%), and total organic matter (TOM, %).Sedimentary types were characterised according to Junoy and Viéitez (1989).Calcium carbonate content (%) was estimated by treating the sample with hydrochloric acid.The total organic matter content (TOM, %) was estimated from the weight loss on combustion at 450ºC for 4 hours.Line drawings of leptostracans were done with the aid of a camera lucida connected to a compound microscope.Drawings of Nebalia reboredae n. sp. were made from the holotype and two dissected adult females.Specimens used for scanning electron microscope (SEM) were dehydrated via a graded ethanol series, prepared by critical-point drying using CO 2 , coated with gold in a BAL-TEC SCD 004 evaporator, and examined and photographed with a JEOL JSM-6400 scanning electron microscope at the Servicios de Apoio á Investigación, Universidade da Coruña (SAIN), Spain.Measurements were made with an ocular micrometer.Total length (TL) was measured from the articulation between rostrum and carapace to the posterior end of the uropods excluding setation; dorsal carapace length (DCL) was considered as the distance between the articulation of the rostrum and the margin of the posterodorsal cleft; lateral carapace length (LCL) was considered as the distance along the lateral surface between the anteriormost and posteriormost margin; carapace height (CH) was measured between the dorsal and ventral margin; rostrum length (RL) was measured along the midline.Description of specimens followed the model of Dahl (1985) and other recent studies (e.g.Martin et al., 1996;Olesen, 1999;Haney and Martin, 2005).The type series of Nebalia reboredae n. sp. is deposited in the Museo Nacional de Ciencias Naturales, Madrid, Spain (MNCN) and in the Zoological Museum Hamburg, Hamburg, Germany (ZMH).
Compound eye: Oval, with ommatidial part covering about two-thirds of total length of eyestalk, dark pigmentation covering almost entire ommatidial part (Fig. 2B).Eyestalk not lobed; ventrodistal region bearing about 30 simple small setae bearing small setules along distal half (Fig. 8A), surface coarsely granulate.Supraorbital scale tapering distally, about half-length of eyestalk.
Antennule: Peduncle four-segmented (Fig. 2E).First article shorter than eyestalk.Second article widest at midpoint, with (1) plumose seta arising from anterior third, (2) cluster of three simple setae and four plumose setae arising from lateral surface, (3) subterminal cluster of about 12 short and long simple setae.Third article shorter than the second, widest distally, with cluster of about eight simple setae on superior distal margin, one long simple seta on inferior distal margin.Fourth article shorter than third, with: (1) medial row of 4-6 simple setae (Fig. 8B); (2) one thick short spine distally; (3) lateral row of 3-4 simple setae; and (4) simple seta arising from inferior distal margin, longer than antennular scale.Antennular scale oval, slightly more than twice as long as wide; anterior margin convex, with: (1) row of setae provided with coarse teeth along margin of distal half (Fig. 8C); (2) numerous simple setae of different lengths with sharper teeth than those of (1) (Fig. 8D); and (3) one long simple seta arising from inferior distal margin.Flagellum well-developed, with 7-8 articles, slightly shorter than the peduncle, each article with: (1) 3-5 aesthetascs as long as or longer than the article; and (2) four simple setae on anterodistal margin, one longer than the others, oriented posteriorly.
Antenna: Peduncle three-segmented (Fig. 2F).First article with anterodistal process ending in one small acute spine.Second article with distal coarsely pointed process.Third article longer than second, with several rows of setae along medial anterior margin: (1) one thin seta and six spine-like setae shorter and thicker than those of (3); (2) 8-9 longer simple setae, the two distalmost associated with four simple setae (Figs.1A, 8E); (3) seven thick spines along proximal half, the distalmost being the longest; (4) five sparsely plumose setae; and (5) a terminal row of 7 spines, increasing distally in length, long plumose seta on posterior margin, cluster of 9 plumose setae and three shorter simple setae along distal interior margin.Flagellum well-developed, composed of 8-9 articles, each article with four terminal setae denticulated at distal end, one shorter than the others (Figs.2G,H, 8F).
Mandible: Incisor process with sharp teeth along inner border, ending in acute process.Mandibular palp three-segmented (Fig. 3B); second article about 1.6 times as long as first; two simple setae: (1) one at mid-length on lateral face, distally plumose; and (2) shorter simple setae subterminally on anterior margin.Third article slightly longer than second, with margins more or less parallel; proximal half of superior margin with row of short setae; inferior margin with three rows of setae: (1) plumose setae from ending of proximal quarter extending to first half; (2) longer plumose setae than those of (1) extending along distal half (Fig. 9A); and (3) about seven curved, dentate setae along distal margin (Fig. 9B).
First maxilla: First endite (proximal) with rounded medial margin bearing simple plumose setae (Fig. 3C).Second endite (distal) larger than first (Figs.3D, 9C); medial margin with two plumose setae and three rows of setae: (1) about 10 setae of increasing size with three large teeth along distal inferior margin accompanied by several smaller teeth (Fig. 9D); (2) about 7 spatulate setae (Fig. 9E); and (3) row of about six setae with several teeth along distal superior margin.Palp well-developed, about five times the length of protopod, with long and spaced setae along its entire length; setae with small setules along their length, apex recurved distally.
Second maxilla: Protopod with four endites bearing setae with setules, endites 1 and 3 being the largest and endite 4 being the smallest (Fig. 3A).Fourth endite with five plumose setae, the distalmost being the longest.Endopod two-segmented, longer than exopod, proximal article 1.4 times as long as distal one; lateral margin with spaced plumose setae; second article with three distal terminal setae, one of them longer than endopod.Exopod longer than the proximal article of endopod; medial margin with plumose setae, two distalmost setae as long as or slightly longer than exopod.
Exopod about 0.7 times as long as protopod; with row of about 12-15 short serrate spines along lateral border ('spine-row'), each spine with tridentate tip, central tooth bifid (Fig. 9F); four stout smooth spines on distolateral border, the distalmost being the longest; long plumose setae along medial margin.
Pleopods 2-4 similar.Protopod with several simple setae on anterior and posterior proximal borders, cluster of two long setae subdistally, short seta next to appendix interna (Figs.6C,D,F); acute triangular process between exopod and endopod, spine next to exopod base; posterior margin even.Endopod longer than exopod, two-segmented; proximal article short, provided with appendix interna; lateral and medial borders of distal article each with about 10 plumose setae, ending in one long spine.Exopod without a row of stout spines, instead with a row of 4-5 spine pairs, each pair consisting of one long and one shorter spine, with short plumose seta in between; medial margin with long plumose setae, three distal spines, the terminal one being the longest.Fourth pleopod protopod with two simple setae and one spine along posterior border on proximal third (Fig. 6E); posterior border even, posterior corner slightly pointed.
Anal somite, anal plates and uropods: Anal somite (pleonite 8) short, slightly longer than pleonite 7. Anal plates with broad bases and acutely tapering distally (Fig. 7B).Uropods elongate, as long as pleonite 7 and anal somite combined.Each uropod with about 16-19 robust setae along lateral margin increasing distally in size, the terminal about 1.4 times length of uropod (Fig. 7A); about 10-12 robust setae and 12-14 plumose setae on lateral inner margin, cluster of three simple spine-like setae and two thin setae subterminally.
Colour: Fixed animals whitish or transparent with apparent dark pigment in eyes.Male.Subadult males have more articles in antennal flagellum (>12) than females, otherwise similar to females.

Morphological variation.
Examination of all specimens in the type series shows that the number of articles of antennular flagellum increases with size (3-6 articles in juveniles of 1.4-2.4mm TL vs 7-8 in adult females of 4.0-4.8mm TL).Juveniles have fewer articles in antennal flagellum than adults (4-5 vs 8-9, respectively).Juveniles have fourth pleopod protopod with posterior corner more acute than that of adult females.The number of spines of pleopod 1 spine-row is smaller in juveniles than in adult females (5-9 vs 12-15).
Ecology.This species was collected from a site at 6.4 m depth whose sediment is medium sand (medi- Distribution.To date, Nebalia reboredae n. sp. is only known from shallow waters of the Ría de Ferrol, Galicia, Spain. Derivatio nominis.The new species is dedicated to Dr. P. Reboreda, a Galician specialist on Isopoda, for her friendship and contributions to the knowledge of taxonomy and ecology of Crustacea from the Galician coast.Remarks.Nebalia reboredae n. sp.differs from the previously described species of Nebalia in the following unique combination of characters: the rostrum is about 2.2 times as long as wide; the antennular scale is slightly more than twice as long as wide; the fourth article of the antennule has up to one short, thick distal spine; the spines forming a row on the external lateral face of the third article of the antenna are similar to each other in length and thickness; the first article of the endopod of the second maxilla is 1.4 times as long as the second one; the exopod of the second maxilla is longer than the first article of the endopod; the posterior dorsal and lateral borders of pleonites 5-7 are provided with distally rounded to truncated denticles; the protopod of pleopod 4 has an even posterior border, with the posterolateral corner slightly pointed; the uropods are as long as pleonite 7 and the anal somite combined; and the terminal seta of each caudal ramus is about 1.4 times the length of the entire ramus.The following species of Nebalia also possess denticles on the posterior dorsal borders of pleonites 6-7 which are distally rounded or truncated: N. bipes (Fabricius, 1780), N. herbstii Leach, 1814, N. clausi Dahl, 1985, N. kensleyi Haney and Martin, 2005, N. kocatasi Moreira, Koçak and Katagan, 2007, from the northern hemisphere, and N. longicornis Thomson, 1879, N. capensis Barnard, 1914, N. ilheoensis Kensley, 1976, N. cannoni Dahl, 1990, N. falklandensis Dahl, 1990, andN. patagonica Dahl, 1990, from the southern hemisphere.Nebalia reboredae n. sp.mainly differs from N. longicornis, N. cannoni, N. falklandensis and N. patagonica in lacking an eye dorsal papilla.In addition, N. bipes, N. herbstii, N. clausi, N. ilheoensis, N. kensleyi and N. kocatasi have a fourth article of the antennule which is provided with 3 or more thick short spines instead of one, and the antennular flagellum bears 10 or more articles in the former species while in N. reboredae n. sp. it bears 7-8 articles.
Nebalia capensis, from southern Africa, resembles N. reboredae n. sp. in its body appearance, in having a similar number of articles in the antennular flagellum and in the shape of denticles on pleonites 6-7.Nebalia reboredae n. sp.differs, however, from N. capensis in having uropods that are as long as pleonite 7 and the anal somite instead of being clearly shorter, an antennal flagellum that bears up to 9 articles instead of up to 11 and a posterolateral border of the fourth pleonite which is not so acutely pointed as that of N. capensis.Furthermore, in N. reboredae n. sp. the rostrum is 2.2 times as long as wide instead of being 2.5 times as long as wide as happens in N. capensis (Barnard, 1914).These two species also seem to differ in the number of short thick spines on the fourth article of the antennule: N. reboredae n. sp. has consistently one spine in all specimens examined instead of 1-2 as happens in N. capensis.Nevertheless, there are discrepancies in the descriptions of N. capensis regarding the number of spines and the presence of a dorsal eye papilla.Barnard (1914) originally described N. capensis as having one short spine and lacking a dorsal eye papilla, whereas Kensley (1976) described specimens from southern Africa assigned to the former species as those having either one spine and one dorsal papilla or two spines and lacking any dorsal papilla.Dahl (1990) considered the presence of a dorsal eye papilla as a highly valuable diagnostic character at species level.Therefore, we suspect that the specimens described by Kensley (1976) might actually belong to two different species.
Nebalia reboredae n. sp. also seems to differ from most of the species of the genus regarding its habitat.Many species of Nebalia have been thought to be associated with conditions of organic enrichment, close to sewage outlets or attracted to carrion (e.g.Barnard (1914), Citarella (1965), Wägele (1983), Rainer and Unsworth (1991), Olesen (1999), Macleod et al., 2007, among others).On the other hand, N. reboredae n. sp. has only been found in oligotrophic sandy sediment in shallow waters, similarly to the conditions observed for other small-sized species (<5.0 mm TL), such as N. daytoni Vetter, 1996, from California (1996a, 1996b, 1996d).On the other hand, in the Ría de Ferrol, N. reboredae n. sp.coexists with N. troncosoi in medium sand bottoms, but the latter is more abundant in fine sand, muddy sand and mainly in Z. marina beds (Moreira et al., 2003a).
Remarks.The examination of the third article of the antennal peduncle has revealed that several long setae located along the medial border and distal end are coarsely serrated along the inferior margin at the distal third (Fig. 1C).This feature was not reported in the original description by Moreira et al. (2003b), nor is it present in the other species reported here.Moreira, Gestoso and Troncoso, 2003 (Fig. 1F) Sarsinebalia urgorrii Moreira et al., 2003b: 200, Figs. 8-14 Ecology.Coarse sandy sediments, from very coarse sand to medium sand, at depths of 12.7-20.0m.Distribution.Galicia, NW Iberian Peninsula (Moreira et al., 2003b).

DISCUSSION
To date, the Ría de Ferrol has the largest number of leptostracan species (6) ever reported from a single area and within a short bathymetric range (<40 m).Another geographical area with comparable leptostracan diversity is California (Pacific coast of North America), from which four new species of Nebalia have been described in recent years (Martin et al., 1996;Vetter, 1996d;Haney andMartin, 2000, 2005).Nevertheless, as our knowledge on taxonomy, distribution and ecology of leptostracans is rather incomplete, it would not be surprising if similar or greater diversity were found in other parts of the world.Extensive sampling of different substrata both rocky and sedimentary will undoubtedly provide further support for this.For instance, Olesen (1999) and Haney and Martin (2000) pointed out the presence of several undescribed leptostracan taxa from the Tanzanian coast and Friday Harbor (Pacific coast of U.S.A.), respectively.Furthermore, Vetter (1996cVetter ( , 1996d) ) found three species of Nebalia (one of them yet undescribed) coexisting in the same area in southern California but only two of them in the same habitat; those species also showed differences in behaviour and life history.In fact, differences in habitat preference are known to occur among species of the same genus within other benthic taxa such as polychaetes, bivalves and amphipods.
Previous work suggests that some leptostracans exhibit preferences for some type of sediment, or rather the inability to survive in other sediments.For example, Vetter (1996c) showed through experimental manipulations that N. daytoni and N. hessleri Martin, Cash-Clark and Vetter, 1996 avoided the habitat of the other species and failed to survive when transplanted to each other's habitat.Nevertheless, the processes which might explain these observations are not known (Vetter 1996c).In the Ría de Ferrol, leptostracans appeared in a number of sediments ranging from gravel to fine sand.No leptostracan was collected in muddy sediments.Furthermore, species of Nebalia were found in a wider range of sediments than those of Sarsinebalia.Nevertheless, previous data show that species of Nebalia in Galician waters tend to appear in greater numbers when seagrasses are present.Moreira et al. (2003a) reported N. troncosoi from medium sand to sandy mud in two inlets in southern Galicia (Ensenada de Baiona and Ensenada do Grove), although this species was more abundant in muddy sand covered by dense beds of the seagrass Zostera marina (L.).In other nearby areas in Galicia, N. strausi was almost exclusively found on Z. marina beds, appearing in smaller numbers on bare muddy sediments (Moreira et al., 2004).Similarly, N. kocatasi and N. strausi have been found in the eastern Mediterranean on muddy sediments mainly with the seagrass Posidonia oceanica (L.) Delile (Koçak and Katagan, 2006;Koçak et al., 2007;Moreira et al., 2007).In the Ría de Ferrol, N. troncosoi and N. kocatasi were found on fine sand with scattered mats of Z. marina, although they were also present in coarser sediments.In addition, N. strausi was only found on coarse sediments, which contrasts with the abovementioned work.However, to our knowledge, in the Ría de Ferrol dense Zostera beds such as those found in other Galician Rías are not present and therefore large numbers of N. strausi are not expected to occur in that ria.On the other hand, the two species of Sarsinebalia found in the Ría de Ferrol were solely collected in gravel or coarser sandy sediments, which agrees with preliminary observations by Moreira et al. (2003b).This strongly suggests that these species might prefer oligotrophic coarse sediments rather than other sediments, particularly S. cristoboi (Moreira et al., 2003b).This species might be locally abundant in the Ría de Ferrol (J.Moreira, pers.obs.) on gravel and coarse sand with shell fragments, and has not been found on sediments of finer granulometry in the Galician Rías yet.Nevertheless, sampling in the Ría de Ferrol was done with different equipment and within several sampling programmes which makes it difficult to establish reliable comparisons of species abundance among different types of sediments.Therefore, extensive quantitative data are needed in order to test the patterns suggested by our results.Manipulative experiments will also help to determine whether leptostracans from the Galician Rías show preference for any given type of substrate.
Species of Nebalia are morphologically very similar to each other and some characters are known to show a certain degree of variation with age and sexual condition.In fact, species from distant areas of the world show many similarities in the structure of mouthparts, appendages and body appearance (Haney and Martin, 2000).Here, we provide a key for the identification of mature females of leptostracans reported from the Iberian Peninsula as well as two further species likely to occur there, namely N. herbstii and N. clausi.The key is mostly based on features of their antenna, antennule, relative length of endopod articles of the second maxilla and dentition of pleonites.Furthermore, a figure is included showing the armature of the third article of the antenna for all species of the Ría de Ferrol (Fig. 1).Although the number of spines and setae may vary depending on age, their shape and length consistently differ among species and can be useful to identify species.In N. troncosoi and N. reboredae n. sp.spines of row 1 have a similar length and thickness while spines of row 3 are shorter and thicker in N. reboredae n. sp.than in N. troncosoi.On the other hand, in N. strausi and N. kocatasi the 3-4 proximal setae of row 1 are thinner and longer than the others, the latter being more robust, particularly in N. kocatasi.All spines are similar within row 1 for both species of Sarsinebalia but those of S. cristoboi are shorter and thicker than in S. urgorrii; spines of row 2 in S. cristoboi seem coarsely serrated along the inferior margin at the distal third instead of being smooth as appears in S. urgorrii.

Table 1 .
-Coordinates and physical characteristics of the sampling sites at which leptostracans were found in the Ría de Ferrol.Q 50 , median grain size; CO 3 = , carbonate content; TOM, total organic matter content; NDR, naturalist dredge; VV, van Veen grab.

Table 2 .
-Comparative table showing collection sites, depth range and sedimentary features for the six known species of leptostracans from the Ría de Ferrol. -8.