Description of the zoeal stages of Periclimenes aegylios Grippa & d'Udekem d'Acoz, 1996 (Crustacea: Decapoda: Palaemonidae) reared in the laboratory

The eight zoeal stages of the Mediterranean shrimp Periclimenes aegylios are described and illustrated in detail, using laboratory-reared specimens. This study improved the partial and unpublished descriptions of the zoeae of this species. The complete and accurate definition of the morphology of all the stages now allows comparison of the zoeae of P. aegylios with those of other Mediterranean Periclimenes species, as well as the allopatric Atlantic P. sagittifer , from which P. aegylios was separated in 1996.


Introduction
Although recent restriction and reviews refer to the genus Periclimenes O.G. Costa, 1884 as a polyphyletic taxon (Bruce et al. 2005;Bruce 2007), it remains a particularly speciose genus of Palaemonidae (Crustacea: Decapoda), with over 155 species (Bruce 2007;Li 2009), being widely distributed throughout the oceans. Some species are reported as free-living, whereas other species are found in association with a variety of invertebrate taxa, including sponges, cnidarians, molluscs, or echinoderms.
Knowledge regarding the lifespan and detailed morphology of larval stages of a species, achievable only through studying reared specimens, is an important tool for understanding the life history of the adult. Larval descriptors are often considered when defining phylogenetic relationships (Ng & Clark 2000;Felder & Martin 2003;Anger 2006), particularly if the adults are difficult to distinguish (Ng & Clark 2000;Cuesta & Anger 2001). Moreover, the morphological description of larval stages of decapods born in laboratory is an important tool to identify planktonic zoeae.

Discussion
The well-known division of the family Palaemonidae into Palaemoninae and Pontoniinae subfamilies was recently questioned by De Grave et al. (2015) who, through molecular analysis, demonstrated that the two taxa do not form clear monophyletic clades. In order to better compare data regarding larval stages obtained from the literature, we continue to use this distinction. Fincham and Figueras (1986) highlighted the differences among the zoeal stages of the two subfamilies: "Palaemoninae" zoeae show "body straight or abdomen gently curved ventrally" and rostrum longer than the half of the antennular peduncle, whereas in "Pontoniinae" the body has "double bend, rather weakly developed in stage 1" and the rostrum reaches about half length of the antennular peduncle (in stage 1). Moreover, according to dos Santos and Gonzáles-Gordillo (2004), the "Pontoniinae" larva is recognizable thanks to "the dorsal connection between carapace and abdomen" forming a right angle. The features, which allow recognizing the larvae of the "Palaemoninae" species, are well described by Fincham and Figueras (1986). As regards Mediterranean "Pontoniinae", at least the first zoea has been described for six species: Ascidonia flavomaculata (Heller, 1864) (Costanzo et al. 1996), Periclimenes aegylios (present work), Periclimenes amethysteus (Geiselbrecht & Melzer 2009), Periclimenes scriptus (Basile et al. 2005), Pontonia pinnophylax (Otto, 1821) (Calafiore et al. 1991), Typton spongicola O.G. Costa, 1844 (Lebour 1925). Therefore, it is possible to arrange a key to distinguish the first larval stage of the above-mentioned species, taking into account features of antennule (A1), antenna (A2), maxillule (Mxl), maxilla (Mx) and maxillipeds 1-2 (Mxp1-2):  Points 4 and 5 of the key suggest that the number of distal segments of antennal exopod is sufficient to distinguish among the Mediterranean Periclimenes species whose larvae are known. Barnich (1996) attributed some general features to Mediterranean Periclimenes spp. larvae: "S-shaped body, eyes looking sideways, pterigostomial spines present, carapace margin not serrated, supraorbital spines long and serrated on ventral side"; from zoea IV, "rostrum with rostral tooth, the tooth being serrated on ventral side, pereiopod 5 without exopod, abdominal somites 1-5 with pleopods, telson in young stages triangular, only very slightly invaginated". Nevertheless, in P. aegylios zoea I the typical S-shaped body is scarcely visible. Furthermore, supraorbital spines appear from the second stage, whereas pleopods start to develop from the sixth stage. The absence of the S-shaped body and the supraorbital spines in the first zoeal stage also occurs in P. amethysteus (Geiselbrecht & Melzer 2009) and P. sagittifer (dos . Regarding the possible comparison among zoeae II-VIII of P. aegylios (present work) and Periclimenes sp. collected from the plankton by Bourdillon-Casanova (1960), it is useful to note that the Author highlighted that the stages II-VII she described could be ascribable both to P. amethysteus and P. scriptus; only the eighth stage resembles the adult of the second species. Even though the present comparison does not concern all the features, because the Bourdillon-Casanova's description (1960) is not complete, the main features of the distinction between the two species are summarized in an additional file [see Additional file 1]. In general, there are widespread differences. Periclimenes larvae coming from Marseille are bigger than P. aegylios ones; size gap is particularly noticeable in the last stage, which is just over 1.5 times longer than P. aegylios. Another distinctive feature is the presence of antennal spines from the third stage, that are always absent in P. aegylios. Other differences regard antennular peduncle, maxillipeds, uropods and telson formula [see Additional file 1]. Moreover, Marseille zoeae show a precocious development of pereiopods and pleopods. Differences in size and development could be ascribable also to the different sources of the specimens under comparison, plankton samples (P. sp.) and laboratory-reared material (P. aegylios).
According to Grippa and d'Udekem d'Acoz (1996), the adults of P. aegylios and P. sagittifer form the sagittifer complex. Regarding the zoeae of this complex, almost every stage shows remarkable differences between the two species, also evident without dissecting the specimens ( Table 1).
Here too, P. aegylios larvae are smaller and show a general retard mainly as regards the development of pereiopods and pleopods, comparing to P. sagittifer. Nevertheless, since the first zoea the differences between the two species are appreciable: zoea I of P. aegylios can be identified from P. sagittifer one because it does not present a distal process on antennular peduncle, it has 5 distal segments (instead of 4) and 9 internal plumose setae (instead of 10) on antennal scaphocerite; the endopod of the first maxilliped is 3-segmented (instead of 4-segmented) with a proximal simple seta on the last segment (absent in P. sagittifer); finally, exopod of maxilliped 2 has 2 proximal simple setae (instead of 1 plumose). The differences between the two species go on throughout the zoeal development; since it is problematic to summarize all the differences of all the zoeal stages, characters are listed in Additional file 2, which gives a detailed and readable comparison. It is interesting to highlight two aspects of these differences. The first one is the segmentation of almost all the appendages. Indeed, in P. aegylios maxillipeds 1 and 2 endopods appear and remain 3-segmented, while in P. sagittifer maxilliped 1 endopod appears 4-segmented and maxilliped 2 endopod becomes 4-segmented from zoea IV. Maxilliped 3 endopod remains 4-segmented since zoea II in P. aegylios, whereas it becomes 5-segmented from zoea V in P. sagittifer. Pereiopods 1 and 2 endopods are 4segmented (5-segmented in P. sagittifer) until zoea VI, whereas pereiopod 3 endopod is 4-segmented (5-segmented in P. sagittifer) until zoea VII. The second aspect regards the development of some characters. In P. aegylios, pereiopod 1 and 2 start to develop the chela in zoea VII (zoea VI in P. sagittifer); pereiopods 3 and 4 endopods appear respectively in zoea III and V (zoea II in P. sagittifer), pereiopod 5 appears in zoea V (zoea III in P. sagittifer). Pleopods development is also shifted: in P. aegylios pleopod buds appear in zoea VI, becoming biramous in zoea VII and developing protopod and appendix interna (in pleopods 2 to 4) in the last zoea, whereas in P. sagittifer pleopods develop one stage earlier, starting from zoea V. Features persistently different throughout the development are, e.g., the presence of a distal process on maxillular endopod of P. sagittifer, which could be detected only in the first zoea of P. aegylios, and the bases of pereiopods 4 and 5, which are naked in P. aegylios while with one seta in P. sagittifer. Moreover, the zoeae of the two species show a different uropods setation. Finally, telson of the two species has the same setal formula in the first three stages and in the last one: P. aegylios zoeae IV to VII does not show the usual developmental pattern of setae, found instead in P. sagittifer.

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The clear morphological distinction detected in larvae supports the separation of the two sagittifer complex species, which was based until now only on the different adults coloration patterns (Grippa & d'Acoz 1996). In order to better understand the meaning and importance of these above-mentioned differences, larval development of the remaining Mediterranean species of the genus Periclimenes should be investigated.