Identification of Lessepsian fish species using the sagittal otolith

Lessepsian species are marine organisms that enter the Mediterranean through the Suez Canal, a phenomenon that has dramatically increased in recent decades. The present study describes the morphological characteristics of the sagittal otoliths of 22 Lessepsian fish species collected at four locations in the Mediterranean Sea. These structures are commonly used in the identification of species found in the digestive tracts of predators, and their morphological description is mainly needed as a tool for trophic studies. Here we used the Automated Taxon Identification (ATI) system of the AFORO web database to determine the accuracy of classifying Lessepsian fish otoliths compared with otoliths of native Mediterranean species. The otolith contour analysis correctly classified 92.5% of the specimens, showing that these species have otoliths that can be clearly distinguished from native ones. Four different groups of otoliths were identified according to the morphological differences between the otoliths and the ATI analysis results: a) unusual shapes with no similarities to native species; b) unusual shapes with similarities to phylogenetically distant native species; c) common shapes with similarities to phylogenetically close native species; and d) common shapes with morphological characters related to other native congeners, although they are not classified with them.


INTRODUCTION
The invasion of Red Sea organisms through the Suez Canal, known as 'Lessepsian migration' (Por 1978) is today a major driver of change in the Mediterranean Sea.Due to the growing importance of this phenomenon, in terms of the number of introductions, successful establishments and geographical expansions (Golani 2010, Zenetos et al. 2010), much attention is now being focussed on Lessepsian species.According to the most recent estimate (Zenetos et al. 2010), around one thousand immigrant species have been recorded so far in the Mediterranean basin.Out of these, almost 60% come from the Suez Canal.The Mediterranean is therefore undergoing a tremendous increase in species richness due to the influx of Red Sea organisms.Hence, basic information about these new comers is urgently needed.
A total of 149 alien fish species have been recorded so far in the Mediterranean Sea and most of them are of Lessepsian origin (Zenetos et al. 2010).They are generally thermophilic species (Azzurro 2008), and it is very probable that the rising seawater temperature is giving them a distinct advantage over native temperate taxa (Galil 2007).The little information available on the biology and ecology of Lessepsian fish is mostly limited to certain abundant and commercially important families, such as Upeneidae and Siganidae (reviewed by Golani 2010).Some work has been dedicated to the trophic relationships of these species in their new environment (e.g.Golani 1993, 1994, Bariche 2006, Azzurro et al. 2007, Zouari-Ktari et al. 2008).Lessepsian fish can also be important prey (Aronov and Goren 2008) but research into their roles in the trophic network is extremely limited.
A fundamental pre-requisite for trophic studies is to be able to identify fish in the digestive tract of other fish based on the morphology of the sagittal otolith (e.g.Pierce and Boyle 1991, Granadeiro and Silva 2000, Moreno-López et al. 2002, García-Rodriguez and De La Cruz-Aguero 2011).The otolith shape has a high degree of inter-specific variation (Nolf 1985), and in most species there is low intra-specific variability.Otolith collections and atlases (Sanz-Echeverría 1926, Nolf 1985, Härkönen 1986, Smale et al. 1995, Rivaton and Bourret 1999, Volpedo and Echeverria 2000, Assis 2003, 2004, Campana 2004, Reichenbacher et al. 2007, Tuset et al. 2008) can be used to identify fish, but more recently the web AFORO (http://www.cmima.csic.es/aforo/)allows automated taxon identification (ATI) based on a database of 950 species and 3300 images of sagittal otoliths.ATI is based on iterative classification using the Euclidean distances of the compared wavelet multiscale descriptors of otolith outlines (Parisi-Baradad et al. 2010).Therefore, each otolith is tested against all the other otoliths in the database.However, the description of the otolith is still relevant because morphological characteristics other than the outline can also help to identify the species, such as the type of sulcus, ostium and cauda (Tuset et al. 2008).Therefore, the main goals of this study were (i) to provide a morphological description of the otoliths of Lessepsian fish, (ii) to compare the otolith morphology of Lessepsian and native species, and (iii) to use the otolith contour to determine the accuracy of the AFORO web as a tool for species identification.

Sampling and data collection
A total of 22 Lessepsian species (18 families and 8 orders) inclusive of the most succesful taxa were collected at four different locations of the central and eastern Mediterranean (Table 1; Fig. 1): 1) Linosa island, Italy (35°51′N 12°51′E); 2) Alexandria, Egypt (31°19′N 30°03′E); 3) Haifa, Israel (32°47′N 34°56′E); and 4) Bozyayi, Turkey (36°5′N 32°59′E).Fish were obtained with the voluntary collaboration of local fishermen, who mainly use trammel nets and trawling.The classification system used was based on Nelson (2006) and the species were named following the criteria of the online fish catalogue of the California Academy of Science (Eschmeyer and Fricke 2011).

Otolith morphology
Images of the left otolith were taken with a digital camera under a binocular microscope.The image was taken of the internal side (medial or proximal) of the otolith because this side has the sulcus acusticus (a groove along the surface of the sagitta).To obtain a clear sagittal contour, the image must be well contrasted with a homogeneous black background.The otoliths were always represented with the respective dorsal margin at the top of the image and the anterior (rostral) region to the right.The terms outlined in Tuset et al. (2008) were used for the morphological description.The following anatomical features were considered: otolith shape, type of sulcus acusticus, shape and size of ostium and cauda, and the characteristics of the anterior and posterior regions (Fig. 2).

Automated identification
Automated taxon identification (ATI) uses wavelet descriptors to extract specific characters of the orthogonal projection of a standard oriented left sagitta according to the methodology given by Parisi- Baradad et al. (2005) and Parisi- Baradad et al. (2010).A multiscale signal representation is obtained from the wavelets.Outline singularities can be located more accurately with wavelets than with Fourier analyses (Mallat 1989).The recognition system searches the AFORO database iteratively, from the coarsest to the finest wavelet scale, to find the otoliths that most resemble the tested otolith.In each iteration, the approximation signal of the tested otolith is compared with each otolith of the database using a Euclidean distance (ED) (Parisi-Baradad et al. 2005, 2010).The otoliths were ordered according to their decreasing similarity to the specimen test, measured as ED.

DISCUSSION
The Lessepsian phenomenon, once limited to the eastern sectors of the Mediterranean (Por 1978), is today a matter of concern for the entire basin.In the last two decades it has increased dramatically, as is evidenced by the increase in the number of invaders and their fast geographical expansion (Golani 2010, Zenetos et al. 2010).Species such as Siganus luridus and Fistularia commersonii have expanded rapidly in the central and western Mediterranean.The latter is now considered well established throughout the entire Mediterranean Sea, as it has been recorded in western areas (Sánchez-Tocino et al. 2007) on the opposite side to the Suez Canal where it originally entered.However, these species have the most impact in the eastern basin.Golani (2010) counted a total of 73 species, which is 14.9% of all the fish known for this sector.Therefore, considering the number of new that have entered the Mediterranean, can hypothesized that the morphological and taxonomic diversity of Mediterranean fish communities has increased.Our range of species includes the more abundant and widespread Lessepsian fish in the Mediterranean Sea, such as Siganus luridus, S. rivulatus, Fistularia comersonii, Upeneus moluccensis, U. pori, Scomberomorus commerson, and Stephanolepis diaspros.Some recent colonists, such as Apogon smithi (Golani et al. 2008) and Plotosus lineatus (Golani et al. 2002), were also considered together with occasional species such as Pteragogus pelycus Randal, 1981 (Golani and Sonin 1992).
Our morphological description of Lessepsian fish otoliths provides new and valuable information for these species.It is also a robust tool for species classification, and agrees in most cases with the ATI system of AFORO.The percentage of correct classifications (92.5%) obtained in this study is greater than that (72%) obtained by Parisi-Baradad et al. (2010) in a study that used the same methodology but with native species.This difference of more than 20% can be attributed to the high morphological specificity of Lessepsian sagittae in comparison with those of Mediterranean native species.In fact, many of these invaders belong to families or genera that are not represented in the native fish fauna of the Mediterranean Sea.Neverthless, ecomorphological characters of otoliths are generally related to the ecological niche that the species occupies (Lombarte 1992, Arellano et al. 1995, Aguirre and Lombarte 1999, Lombarte and Popper 2004, Ramcharitar et al. 2004, Schulz-Mirbach et al. 2008, Colmenero et al. 2010, Tuset et al. 2010).Thus, the highly differentiated otolithic morphology of Lessepsian species could reflect specific adaptations (Schoener 1974, Motta et al. 1995), and hence novel opportunities to thrive in the newly colonized habitat.This hypothesis recalls the controversial concept of 'niche opportunity' that has often been used to explain the success of these invaders (Por 1978, Lundberg and Golani 1995, Oliverio and Taviani 2003).This concept is not within the scope of the present research, and further ecological studies are necessary to evaluate it.
There is currently little information on the morphology of Lessepsian fish otoliths.Tuset et al. (2008) published an otolith atlas that included an exhaustive description of the sagittae of the most common species inhabiting the Mediterranean Sea, but no Lessepsian species were included.Lessepsian species can be classified into four different groups according to morphological differences between otoliths, ATI analysis and the otolith descriptions of species from the Mediterranean Sea (Tuset et al. 2008): a) Unusual otolith shapes with no similarities to other native species.This is the case of Plotosus lineatus, which is a Siluriform and like other otophysian teleosts has a sagitta that is very different from that of non-otophysian fish (Popper andPlatt 1981, Nolf 1985); Fistularia commersonii, which is a syngnathi-form with a very unusual body shape and size compared with the Syngnathiformes native to the Mediterranean; Atherinomorus forksalii is an atheriniform but does not show any similarities to otoliths from Mediterranean Atherinidae.
b) Unusual otolith shapes with similarities to phylogenetically distant native species.Otoliths of Saurida undosquamis showed similarities to the Sphyraenidae, although this group is characterized by a well-differentiated ostium and cauda; Apogon smithi matched with Coelorinchus caelorhincus (Risso 1810) (Macrouridae) and with the Epigonidae, but the sulcus acusticus and shape were different; Sillago sihama was similar to Gnathophis mystax (Delaroche 1809) (Congridae) and Phycis spp.(Phycidae), although G. mystax has a heterosulcoid sulcus and Phycis spp.has a more lanceolate or spindle-shaped otolith; Sargocentron rubrum had a similar shape to some sparids, especially to Pagellus bogaraveo (Brünnich 1768), although the otolith was easily identified due to its thickness, as occurs in other Holocentridae; Equulites klunzingeri joined with Atherina spp.(Atheriniidae) and Mullus barbatus Linnaeus 1758 (Mullidae); Atherina spp.showed an excisura ostii with or without a shorter notch, while Mullus had a curved sulcus; Terapon puta was classified as Scorpaena spp., although the otolith was characterized by a more acute rostrum, a larger ostium area and a less curved cauda; the otoliths of Pempheris vanicolensis resembled those of Epigonus constanciae (Giglioli 1880) (Epigonidae), which have a curved cauda, a pointed rostrum and an entire dorsal margin without sculpture; the otoliths of Pteragogus pelycus were similar to those of Apogon imberbis (Linnaeus 1758) (Apogonidae) and to the otoliths of Labridae.Nevertheless, they can be easily identified due to the unusual cauda form and the V-shaped ventral margin; Siganus rivulatus was close to species with a double-peaked anterior region, e.g.Sardinella aurita Valenciennes 1847 (Clupleidae), Labrus mixtus Linnaeus 1758 (Labridae) and adult Lichia amia (Linnaeus 1758) (Carangidae).In Clupeidae and Labridae the end cauda is not curved, while L. amia has more irregular margins and a tubular shaped cauda.Finally, Sphyraena chrysotaenia otoliths have a similar shape to those of Serranus cabrilla (Linnaeus 1758) (Serranidae), which can have a shallow notch in the excisura ostii.c) Common otolith shapes with similarities to phylogenetically close native species.The otolith morphology of Etrumeus teres is comparable to that of Sardinella spp.and Sardina spp., and also resembles otoliths of other pelagic species, such as Scomber scombrus Linnaeus 1758 (Scombridae), Trachinotus ovatus Linnaeus 1758 and Caranx rhonchus Geoffroy Saint-Hilaire 1817 (Carangidae).Nevertheless, the antirostrum was less developed, short and rounded, with a shallower notch in the excisura ostii in comparison with other clupeids.It also differed in the shape of the sulcus acusticus and the rostrum and postrostrum regions, which are more pointed in carangids and scombrids.Pomadasys stridens was similar to Pomadasys incisus (Bowdich 1825) and to sparids Spondyliosoma cantharus (Linnaeus 1758).The identifiable feature of P. incisus was an indentation in the anterior-dorsal margin, whilst S. cantharus shows thinner otoliths with a median sulcus acusticus and slightly curved cauda; Upeneus pori resembled Mullus spp., although there were no deep or strong depressions at the end of the cauda; Scomberomorus commerson had a shape characteristic of pelagic species, such as young Seriola dumerili (Risso 1810) (Carangidae), and Scombridae, such as Sarda sarda (Bloch 1793); the otoliths of young S. dumerili have a more peaked rostrum and antirostrum as well as lobulated dorsal margins, and the otoliths of S. sarda have a triangular shape and a more elongated rostrum.Finally, the otholits of Stephanolepis diaspros and Lagocephalus suezensis were easy to identify and comparable with otholits of other Tetraodontiformes, including Sphoeroides spp.d) Common otolith shapes with morphological characters related to other native congeners, although they are not classified with them.Upeneus moluccensis is similar to Mullus spp., Siganus luridus resembles Cupleidae and Labridae because of a double-peaked anterior region, and Lagocephalus sceleratus has an otolith shape that is very representative of Tetraodontidae.
To conclude, the unusual otolith shapes of Lessepsian fish species led to a very high classification success.In addition, the analysis of Lessepsian otoliths provided new morphological information that can be used in species identification with the ATI system, and applied in other scientific fields, such as establishing marine trophic networks (Moreno-López et al. 2002, García-Rodriguez andDe La Cruz-Aguero 2010).Future studies are needed to complete the morphological catalogue of Lessepsian otoliths.This information can be used not only as a descriptive tool, but also to test theories in the ecomorphological field of invasion biology.

Table 1 .
− Taxonomical identification, area and total length range of Lessepsian species studied in the Mediterranean Sea.

Table 2 .
− Comparison of the otolith contour of Lessepsian species with other species from the Mediterranean Sea using the automatic system analysis of AFORO web.ED, Euclidean Distance.