A taxonomic revision helps to clarify differences between the Atlantic invasive Ptilohyale littoralis and the Mediterranean endemic Parhyale plumicornis (Crustacea, Amphipoda)

Abstract Ptilohyale explorator (formerly Parhyale explorator), described by Arresti (1989), can be considered to be a synonym of west-Atlantic Ptilohyale littoralis (Stimpson, 1853), based on morphological observations of paratypes and specimens recently collected in the type locality of Ptilohyale explorator. The first collections of Ptilohyale littoralis, from the eastern Atlantic were from the port of Rotterdam (The Netherlands) in 2009 and later in Wimereux, Opal Coast (France) in 2014; however, the synonymy of Ptilohyale explorator with Ptilohyale littoralis backdates to the first European record of Ptilohyale littoralis in 1985 at La Vigne, Bay of Arcachon (France). This indicates that Ptilohyale littoralis has been established along European Atlantic coast for many years. An assessment of the nominal valid species belonging to the genus Ptilohyale was carried out and a comparison between the Atlantic Ptilohyale littoralis and the very similar Mediterranean hyalid species, Parhyale plumicornis, is presented based on morphological features and distribution. Due to the invasive ability of Ptilohyale littoralis, a comparison between the two species is necessary.


Introduction
Ptilohyale explorator (formerly Parhyale explorator) was described by Arresti (1989) from La Vigne, Bay of Arcachon, France. He collected eleven male and three female specimens in the intertidal zone, on the sand of a semi-enclosed beach under stones, in July 22, 1985; following Barnard's (Barnard 1979: 120) "Key to the Species of Parhyale and Parallorchestes", he established that the specimens sampled showed a feature that was not included in the key, i.e., the presence of long tufts of plumose setae in antenna II starting at the 5 th peduncular segment. However, as Barnard's taxonomic key (Barnard 1979) had omitted some hyalid species already described at that time, Arresti did not take into consideration some preceding descriptions (listed in Table 1) that could fit with his collected specimens.
As a consequence, Arresti described a new hyalid species under the name Parhyale explorator, and deposited eight males and two females in the laboratory of the University of the Basque Country (Spain; holotype; allotype; six males and one female paratypes), one male paratype in the Carcinology Laboratory of Natural History Museum of Paris (France), and one male and one female paratypes in the Laboratory of Dr. S. Ruffo in the Museum of Natural History of Verona (Italy).
In 2008, Ptilohyale explorator (Arresti 1989) was reported as a new alien species within the Mediterranean Sea (Bakir et al. 2008), but later acknowledged to be a misidentification (Bakir et al. 2013), who re-identified the samples as Parhyale plumicornis (Heller, 1886), an endemic Mediterranean species (Iaciofano and Lo Brutto 2017). Regrettably, the case of this erroneous identification caused a cascade-effect on successive papers and documents that reported a further non-indigenous species (NIS) within Mediterranean (Bakir et al. 2010, Christodoulou et al. 2013, Faasse 2014, Zenetos 2010, although this was not the case.
Ptilohyale explorator is currently considered a valid species even if some authors have already highlighted the need of further investigations, in light of its high similarity with Ptilohyale littoralis (Faasse 2014, Spilmont et al. 2016, Marchini and Cardeccia 2017.
To clarify the position of Ptilohyale explorator, here considered species inquirenda, the paratypes deposited at the Natural History Museum of Verona and at the Natural History Museum of Paris were examined, together with some topotypic specimens collected in the type locality of the species, Bay of Arcachon, France. Descriptions and illustrations of current species belonging to the genus Ptilohyale were also consulted, and it was observed that some of them were not ascribable to this genus.

Materials and methods
The paratypes of Parhyale explorator (voucher number 330/P) deposited in Sandro Ruffo's collection of the Museum of Natural History of Verona, Verona (Italy) and the Ptilohyale explorator paratype (voucher number MNHN-Am3957) deposited at  (Barnard 1979: 120) and by Arresti (1989), here named according to  and  the crustaceans collection of the Natural History Museum of Paris (MNHN), Paris (France) were examined under a stereo-microscope, and photos were produced.
Additionally, a total of 126 specimens of Ptilohyale sp. (84 females and 42 males) was collected in the intertidal zone associated with mussel beds (Mytilus edulis), from Bay of Arcachon, France (the type locality of Ptilohyale explorator), 43°34'N, 1°14'W (DDM), in October 2015, and fixed in 95% ethanol. Their body lengths, from tip of rostrum to apex of telson, were measured using ImageJ software after placement on graph-paper and photography (FINEPIX S1800, FUJIFILM); pencil drawings were scanned and 'inked' using the software Adobe Illustrator CS5. The specimens were identified as Ptilohyale littoralis and deposited at the Museum of Zoology of the University of Palermo (MZPA), Palermo (Italy), Voucher Number MZPA-AMPH-0024.
Descriptions of the 12 world species of the genus Ptilohyale, according to the World Amphipoda Database , were consulted and the diagnostic characters delimiting Ptilohyale Bousfield & Hendrycks, 2002 were verified: (1) heavily plumose (finely brush-setose) antenna II starting at the 5 th peduncular article (both sexes); (2) lack of a guiding robust seta on the medial face of the propodus of gnathopod I (male); (3) variously developed carpal lobe of gnathopod II (male); (4) distomedial robust seta on the peduncle of uropod I; (5) inner ramus of uropod III more or less fused to the peduncle. The subsequent generic status for each of these species was then revised.

Results
The paratypes of Ptilohyale explorator preserved in the Museum of Natural History of Verona were entire and in good condition for observations ( Fig. 1), while the paratype stored in the Carcinology Laboratory of Natural History Museum of Paris had deteriorated and some body parts were lost (i.e., heads).
Comparison with the description of Ptilohyale explorator (Arresti 1989: 103-111) and the paratypes stored at the museums of Verona and Paris showed some incongru-  ences ( Table 2). The most significant difference was the absence of a strong depression on the basipodite of peraeopod VII in all paratypes, conversely to what was indicated in the description. Other diagnostic characters described by Arresti (1989) were also unlike the paratypes, including the number of plumose articles on antenna II, and the arrangement of setae on uropods I and III (see Table 2 for details).

Systematics
Type locality. Grand Manan Island (Canada), northern eastern Atlantic coast. Material examined. One hundred and twenty-six specimens were collected at the Bay of Arcachon France (43°34'N, 1°14'W), 13October 2015; intertidally, 0 m, on the heavy substrate of the semi-closed beach (MZPA-AMPH-0024). Description. Male. 11.4 mm length specimen. Antenna II ventral margins of the 5 th peduncular article and first 4-9 flagellar articles (other one or two articles with sparse plumose setae) densely covered with plumose setae (brush setae). Palp of maxilla I with median constriction. Coxal plate I sub-quadrate with distinctive cups; Gnathopod I, basis lacking distinct anterodistal lobe (hydrodynamic lobe). Gnathopod II, carpus lobe present in juvenile male and absent on adult male. Coxal plate V posterior lobe smaller than anterior lobe; Peraeopod V, basis rounded. Peraeopod VII slender, basis rounded. Uropod I, peduncle with one distomedial robust seta; rami subequal with 3-4 robust setae on outer ramous and 1-2 robust setae on inner ramus. Uropod II, rami sub-equal in length. Uropod III, outer ramus with 5-9 apical robust setae. Telson acute. Female. Description based on a 10.6 mm length specimen. Gnathopod I, basis with anterodistal lobe.
Distribution. Northern, western, and eastern Atlantic coasts; north eastern Pacific coast.

Discussion
Ptilohyale (formerly Parhyale) explorator was described by Arresti (1989) using the dichotomous key to "Parhyale and Parallorchestes" of Barnard (1979: 120); he observed that the specimens collected were not ascribable to any of the species listed therein, due to the presence of dense elongate tufts of plumose setae ventrally on the peduncular article 5 of the antenna II and peduncle of uropod I with distomedial robust seta. These characters (and others listed in Table 3) prompted Arresti to describe a new species, and to revise Barnard's key; however, both authors had excluded some hyalid species that could be identified with Arresti's specimens ( Table 1).
The following character states are considered diagnostic of Ptilohyale explorator: the arrangement of setae on the uropods and the presence of a strong depression on the posterior margin of the basis of peraeopod VII (Table 2). Here, it has been verified that these characters described in Arresti (1989) did not match with the paratypes (Fig.  5). The setae arrangement on uropod III and the posterior margin of basis of peraeopod VII of the paratypes, on the contrary, matched with specimens recently sampled from the explorator type locality and were identified as Ptilohyale littoralis. In fact, following the dichotomous key to Hyalidae of Bousfield and Hendrycks (2002), the detailed description of Faasse (2014), the paratypes in Ruffo's collection, and the present specimens collected in Bay of Arcachon can all be identified as Ptilohyale littoralis (Stimpson, 1853). For these reasons, Ptilohyale explorator (Arresti, 1989) is proposed as a synonym of Ptilohyale littoralis (Stimpson, 1853), which, on base of the Principle of Priority, article 23 of the ICZN Code (Ride Chairman et al. 1999), becomes the valid name of this taxon. Bousfield (1973) synonymised Ptilohyale littoralis with Ptilohyale plumulosus, a species distributed along the Pacific coast of North America. This synonymy was subsequently rejected (Bousfield and Hendrycks 2002), thus limiting the distribution of Ptilohyale littoralis to the western Atlantic coast of North America (Bousfield and Hendrycks 2002).
Recently, Ptilohyale littoralis was declared as a recent alien species spreading along the eastern Atlantic coast since 2009 (Faasse 2014, Spilmont et al. 2016, Marchini and Cardeccia 2017, but this study has shown that the species inhabited the Atlanto-European coast at least since 1985. Moreover, Ptilohyale littoralis was recently recorded along the eastern Pacific coast of North America (Campbell River, Vancouver, Choi et al. 2016;and Puget Sound, Washington State, Heerhartz et al. 2016), suggesting an extension of the species' range.
The genus Ptilohyale has been diagnosed with plumose setae on ventral margins of antenna II that start at the 5 th peduncular segment and distomedial robust seta on peduncle of uropod I. Behaviourally, it is described as saltatory and occurring in brackish and estuarine waters (Bousfield and Hendrycks 2002).
Uropod III with apical and dorsal setae; Inner ramous of uropod III well defined and not fused to the peduncle; Uropod I without robust seta on peduncle.
Uropod III with apical and dorsal setae; Propodus of peraeopod VII with setae on posterior margin.

The Atlantic Ptilohyale littoralis vs. the Mediterranean Parhyale plumicornis
Due the high connectivity between eastern Atlantic and Mediterranean area, which has already caused a high similarity in the Portuguese and Mediterranean amphipod fauna (Plicanti et al. 2016) it can be supposed that the Atlantic Ptilohyale littoralis may have spread into the Mediterranean, or vice versa, Parhyale plumicornis into the Atlantic Ocean.
Parhyale plumicornis belongs to the Mediterranean fauna and it is the most similar hyalid species to Ptilohyale littoralis due to the overlapping morphological and ecological characters such as the brush setae along ventral margin of antennae II (Bakir et al. 2013, Iaciofano andLo Brutto 2017); and both their presences in the intertidal habitat in slow-drying sediments (Arresti 1989, Bousfield and Hendrycks 2002, Iaciofano and Lo Brutto 2017. Some morphological character states are presented in Fig. 6 as a guide to the correct identifications: Ptilohyale littoralis (Fig. 6A) has brush setae on the ventral margin of antenna II that start at the 5 th peduncular article (Fig. 6B) and distomedial robust setae on the peduncle of uropod I (Fig. 6C). In contrast, Parhyale plumicornis (Fig. 6E) has brush setae on the ventral margin of antenna II that start at the 4 th peduncular article (Fig. 6F) and has a distolateral robust seta on the peduncle of uropod II (Fig. 6G).
These two species show a different and non-overlapping distributions: Ptilohyale littoralis was recorded along European Atlantic coast ( Fig. 6D; Table 4), whereas Parhyale plumicornis was recorded along Mediterranean and Red Sea coasts ( Fig. 6H; Iaciofano and Lo Brutto 2017 and reference therein). In light of the range extension of Ptilohyale littoralis already recorded over long distances, a clear representation of diagnostic character states is needed. Ptilohyale littoralis may be invading the Mediterranean Sea where it could probably be a competitor of the Mediterranean endemic Parhyale plumicornis as it occupies the same habitat.