Marine Nematodes from the Shallow Subtidal Coast of the Adriatic Sea : Species List and Distribution

is study is the �rst attempt aiming to assess the composition and number of free-living marine nematode species on the coasts of the Marches region, Italy. A high number of putative species of nematodes were recognized (84), these belonging to 52 genera in 22 families. Fiy-one taxa have been identi�ed to the species level increasing the number of known nematode species for the Adriatic Sea from 283 to 310 and for the Mediterranean Sea from 700 to 723. e highest diversity and abundance were registered for the nematode families typically of intertidal zones characterized as medium-�ne sands.emajority of the species found in the present study are known to occur in the North European coasts or the North Atlantic Sea, the best known regions for nematode distribution. Inferences on the biogeography ofmarine nematodes are preliminary sincemost Biodiversity literature concerning the Mediterranean of the basin is very out of date. Considering the great importance of nematodes in the assessment of ecosystemhealth conditions, an intensi�cation of sampling efforts should be pursued in other regions in order to improve our current knowledge of the distribution pattern of marine nematode species as well as clarify their biogeographical patterns.


Introduction
Marine nematodes are the most abundant metazoans in marine sediments, reaching densities as high as 20 million individuals per square meter [1].ey generally are the dominant component of the meiofauna in any aquatic habitat, oen one order of magnitude higher than any other major taxon [1].Estimates of global nematode species diversity have signi�cantly varied over the past 15 years, but with a growing agreement suggesting about one million species [2,3].eir great importance in marine ecology as bioindicators is well recognized [4,5].e study of nematode communities offers several advantages for assessing the quality of freshwater, marine, and terrestrial ecosystems.ese organisms show great diversity and abundance which make easier their sampling; they present different trophic groups as well as being intimately related with their environment, the sediment.erefore, they can provide important information on the ecological state of a given area once they are exposed to pollutants.Furthermore, they have been recently proposed within the Water Framework Directive (WFD, Directive 2000/60/EC) [6] as a tool for evaluating the ecological quality status of marine vulnerable ecosystems.e taxonomy of marine nematodes is known to be very difficult (see for review [7]), in part due to their high diversity.Identi�cation of nematodes at speci�c level is oen hampered by the fact that a signi�cant proportion of the specimens collected are juveniles or females, which oen lack the diagnostic features required for an accurate identi�cation.When males are encountered, it may be necessary to examine a high number of specimens before a �nal decision can be made.Owing to these limitations, biodiversity studies focusing on marine nematodes have been frequently replaced by ecological studies where identi�cation is mostly limited to the genus level (or more rarely morphotypes).Unfortunately, this kind of information may be hardly used to create a map of distribution of the free-living marine nematodes.
A recent review of the state of knowledge of the meiofauna in the Adriatic sea has been carried out by Balsamo et al. [8].Marine nematodes known in the Italian seas represent 443 species which are distributed into 262 genera and 46 families [9]; these are a quarter of the species reported for the International Journal of Biodiversity European sea waters.Although these data are an important base line for the area, most of these records are the result of old surveys, mainly carried out in the �rst part of the XX century [9].In particular, the Adriatic coast poses a high number of nematode species: 283 species in the entire Adriatic Basin and 263 in the Italian coasts (see [8] for review).Extensive investigations in this area were carried out by Allgén [10], Schuurmans-Stekhoven [11], Gerlach [12], and Travizi and Vidaković [13].Most of the species were recorded in the area of the river Po, Venice lagoon, and Gulf of Trieste [13][14][15].A signi�cant contribution to the knowledge of marine nematodes for the southern Adriatic Basin, in particular the Apulian littoral, was provided by De Zio [16,17] and Grimaldi-De Zio [18][19][20].erefore, most of the investigations on nematodes have been limited to the North Adriatic sector whereas from the coast of Emilia-Romagna to Apulia few studies were carried out [9,13].
Despite their great ecological importance [6], the knowledge of the nematodes in the Adriatic has changed a little, except for data reported by some ecological studies where nematode identi�cation is mostly limited to the genus level [21][22][23][24][25][26].
In this context, a nematode survey in the coastline between Fiorenzuola and Pesaro (Marches region) has been carried out.Currently, there is no data available on nematode diversity for this area as well as in the Marches region.Furthermore, this coast includes the Natural Regional Park of Monte San Bartolo, an area of high naturalistic value.us, the goal of the present study was to assess nematode biodiversity along this coastal area as well as to identify patterns of species distribution.

Study Area and Sampling
Sites.e Adriatic sea is an elongated NW/SE oriented basin located in the central Mediterranean Sea and characterized by the most extensive development of continental shelf in the Mediterranean.It is a relatively shallow basin, especially in the northern sector, with an average water depth of 35 m. e general circulation is cyclonic, with a �ow towards the northwest along the eastern side and a return �ow towards the southeast along the western side [27].e central and southern regions of the Adriatic Sea are characterized by a low primary productivity, with the continental inputs and the benthic-pelagic interactions being of minor importance in comparison to the northern area [28].
e investigated area is located in the northern part of the Marches region (Italy) and extends from Fiorenzuola di Focara to Pesaro (Figure 1).e nematode species reported in the present study were collected in three different sites from the sublittoral zone of the Marches coast: Fiorenzuola di Focara (FI) (43 ∘ 57 ′ N-12 ∘ 49 ′ E), Monte Brisighella (BR) (43 ∘ 56 ′ N-12 ∘ 50 ′ E), and Pesaro (P) (43 ∘ 55 ′ N-12 ∘ 53 ′ E) (Figure 1).In each site, two stations were established (1 and 3 m of depth) and sediment samples collected in May and November 2003.In addition, a third station was established at FI site (12 m in depth) which was sampled in May, July, and October 2002.e total of the samples studied for the taxonomical study of the nematodes were 15.

Sample Processing.
Bulk sublittoral samples were taken using a 1.5 L plastic scoop digging it directly into the sediment at shallow sites and using a Van Veen grab at the deeper sites (3 and 12 m).Nematode samples were �rst �xed in a 7% magnesium chloride aqueous solution and then posteriorly using a 4% formaldehyde solution (in a buffered sea-water).Two additional samples were taken to determinate the sedimentological features as well as total organic content of each site, depth, and period.

Abiotic Parameters.
Grain size analysis was performed on the collected samples using a vibrosiever for fractions larger than 63 m and an X-ray analyzer for those smaller than 63 m.Sediments were classi�ed following the Wentworth scale [29].
Total organic matter content was gravimetrically determined aer loss on ignition [30].e sediment samples were �rst dried at 60 ∘ C for 6 h and weighed on a Scaltec SBC21 microbalance (accuracy 0.1 mg) to obtain the dry weight.Samples were then combusted in a muffle furnace (550 ∘ C for 4 h) and reweighed to determine the ash fraction.e organic fraction content was calculated by subtracting the ash weight from the total weight.

Nematode Study.
Nematode specimens were extracted by decantation or by centrifugation through a silica gel gradient (Ludox HS 30, density 1.18 g cm −3 ) [31].About 100 nematode specimens from each sample were picked out under a stereomicroscope, transferred into glycerine, and mounted on permanent slides.Nematode species identi�cation was performed using a light microscope equipped with Nomarski optics (Optiphot-2 Nikon) and aided by the pictorial keys in Platt and Warwick [32,33], Warwick et al. [34], and other relevant literature available on Deprez et al. [35].Genera and species identi�ed in this study are reported in alphabetical order (Table 1).e validity of scienti�c names was veri�ed based on Deprez et al. [35] and WoRMS (http://www.marinespecies.org/).Synonymies species were also consulted in Gerlach and Riemann [36].e habitat features (i.e., depth and sediment type) of the described taxa are indicated by symbols.
Current data on the distribution of Italian marine fauna as well as nematodes species [9] cover the nine biogeographical zones of the Italian sea as suggested by Bianchi [37].In this sense, the area sampled in the present study is also part of the North Adriatic sector.

Results
e granulometric analysis of the sediment samples revealed that all the shallow subtidal sites (1.5-3 m in depth) are characterized as medium-�ne sand (Mz 250 m).ese samples contained a high percentage of sand (96%) very low contents of mud (3%) and gravel (1%).On the other T 1: List of nematode species found along the Marches coast and their distribution in the Mediterranean and Adriatic Seas.Sampling site and habitat/sediment type are reported for each species.

Species
Sampling site Habitat/sediment New records 1,2,3   hand, the sediments of the subtidal site (12 m in depth) were characterized as muddy sediments (Mz 25 m) with a higher mud content (64%), followed by sand (35%) and gravel (1%).e total organic content (TOM) was lower in the shallow subtidal sites (2%) than that in the deeper sites (5%).
Cyatholaimidae were represented by 7 putative species (3 only identi�ed to species) which belong to the following genera: Cyatholaimus, Pomponema, and Praeacanthonchus.

Discussion
e general composition of the nematode fauna found in this study seems related to the habitat and sediment types (i.e., medium-�ne sands) [13].Most of the recorded species are in the shallow sublittoral zones: this may be related both to a more extensive investigation carried out in the shallow waters and/or to a lower nematode richness and diversity found offshore [26].Indeed, the increase in the biodiversity of nematodes in parallel with the size of the sediment is well known [4,39,40].In fact, the sediment grain size appears to be one of the main environmental variables in�uencing the horizontal distribution of nematodes.Different sediment types are characterized by different nematode assemblages and by a predominance of speci�c families, so-called isocommunities or parallel communities (see [4,41,42]).
Although Xyalidae is a family usually predominant in �ne sediments, it may occur (with different ratio) in a wide range of sediment types, from muddy to coarse sands [4,26,40,43].All the Xyalidae specimens identi�ed at speci�c level were found in shallow sites and are typical of intertidal habitats (see [34]) (Table 1).e highest diversity of this family in the shallow waters proves the importance of genera like eristus and Daptonema in this environment, which includes sheltered habitats with medium-to-�ne sands.On the other hand, Comesomatidae representatives are commonly related to muddy sediments, oen characterized by anoxic conditions [4,26,44].Indeed, these species were more diverse and abundant in the offshore station of FI, characterized by sediments with a high content of mud and organic matter.In muddy sediments, this family can be dominant, representing up to 34% of the total nematode community [26].Comesomatidae were mostly represented by species of the genus Sabatieria: S. breviseta, S. pulchra, S. ornata, as well as by Dorylaimopsis mediterranea, S. �bulata, and S. hilarula.Among these species, S. pulchra is known to be physiologically well-adapted to stress conditions (see [39]), being able to survive in de-oxygenated sediments as a facultative anaerobic species [45].However, S. ornata seems to be con�ned to the oxidized sediment layers.Among the species found offshore, Terschellingia longicaudata, T. tardus, Halichoanolaimus dolichurus, O. rectangula, and A. elongatus are typical of �ne sediments rich in silt fraction [34,[46][47][48].However, some of the offshore species were also found in the coastal stations (e.g., O. rectangula, S. breviseta, S. pulchra, S. ornata, and S. �bulata).e specious Oncholaimidae was one of the most widespread and abundant nematode family in the shallow waters, with some species typical of intertidal zone and coarse sands [4,34], while being less representative offshore [26].is might be related to the life strategy of some species within this family, which might be sensible to anoxic conditions found offshore [49].Species, such as O. campylocercoides, might be an exception.Frequently in anoxic sediments, O. campylocercoides and T. longicaudata present sub-epidermic grains that probably represent a detox-i�cation mechanism from the high sul�de concentrations [50,51].As Comesomatidae, Linhomoeidae and Xyalidae are dominant and characteristic of muddy sediments, whereas the importance of Chromadoridae increases with the increasing of median grain size [4,40,48].In fact, Chromadoridae were more frequent and abundant in the shallow sublittoral samples, with species typical of the intertidal zone and sandy substrates.
Biogeographic hypotheses for most of the inconspicuous meiofaunal organisms, and thus also for nematodes, are oen very difficult.is is mainly due to the "taxonomic crisis" that concerns most meiofaunal taxa and also noticeable by the signi�cant decrease of biodiversity surveys in the last decades [8,52].Costello et al. [53], updating the European marine biodiversity inventory, have highlighted that the majority of the nematode reports in the Mediterranean area comes from out of date literature as well as total absence of identi�cation guides for the Mediterranean basin.Furthermore, the guides oen only report the widespread, common, and/or ecologically signi�cant species.In contrast, many of the rarer or taxonomically ambiguous species (oen because of the very poor quality of the original descriptions) are not covered in any guides.A consequence is that the �rst species are much more frequently reported in the faunistic studies and thus present wider distribution, while the latter species appear to be locally distributed.
Overall, the families and genera reported in this study show a wide global distribution, while the species detected are typical of the North Western Europe or the Northern Atlantic; see Deprez et al. [35] and WoRMS [38].is is in part due to the presence of most marine nematode taxonomists in the North Western Europe.In addition, most of the marine nematode literature is concentrated in this region.All these elements must be considered when inferences on nematode distribution and biogeography are discussed.Furthermore, recently Fontaneto and Hortal [54] have recently brought up the question "if microorganisms-freshwater animals lower than 2 mm-can have a biogeography" to the attention of the scienti�c community.e authors argue about the "Everything is Everywhere" (EisE) hypothesis, suggesting that the geographical distribution of these organisms may be strongly in�uenced by their animal size.Indeed, for inconspicuous freshwater organisms, the high potential for dispersal might contribute more in determining a wider distribution than that in the large ones [55].e occurrence of morphologically identical nematode species in completely divergent habitats ("meiofauna paradox" sensu Giere, [56]) is also common in marine meiofauna.However, the existence of cryptic species (a complex of morphologically identical species which can only be differentiated by DNA) or a parallel evolution in response to similar microhabitats seems to be more plausible explanation to this trend than the dispersal capacities (see for review [41]).erefore, theories on the biogeography of nematodes as well as other meiofaunal groups should be proposed with caution.
e present study represents a �rst attempt to increase the knowledge of free-living marine nematodes in the Adriatic basin and the Mediterranean Sea.It aims to establish the basis for a future discussion of the biogeography of marine nematodes in the Mediterranean Sea, currently biased by also dated and fragmentary information.

Conclusions
Biodiversity investigations aim to integrate species checklists and the compilation of databases that represent a regional and global bene�t for researchers worldwide.Furthermore, the monitoring of biodiversity over time is of great importance for planning conservation actions, which seems to be more urgent these days, especially in vulnerable coastal systems.is study represents the �rst survey of the marine nematodes in the Marches coasts.e families with the highest abundance and diversity found in this study are typical of intertidal habitats, especially in sediments characterized by medium-�ne sands.Most nematode species found in this study were previously reported only in the North Western Europe and the North Atlantic which are the most known regions regarding marine nematodes.is shows the necessity of intensifying sampling efforts in other regions to improve the current knowledge of marine nematode distribution as well as to clarify their biogeographic patterns.