Description of a new sepioline species , Sepiola boletzkyi sp . nov . ( Cephalopoda : Sepiolidae ) , from the Aegean Sea

A new sepioline species, Sepiola boletzkyi sp. nov. (Cephalopoda: Sepiolidae), is described based on two specimens from the Aegean Sea (eastern Mediterranean). The type specimens are lodged in the Ege University Faculty of Fisheries Museum of Izmir (Turkey). The new species belongs to the Sepiola atlantica group sensu Naef, hence it is compared with the species in this group, namely Sepiola affi nis, Sepiola atlantica, Sepiola bursadhaesa, Sepiola intermedia, Sepiola robusta, Sepiola rondeletii, Sepiola steenstrupiana and Sepiola tridens. The male of S. boletzkyi sp. nov. differs from all the others in having the combination of homomorphous ventral arm tips, eight enlarged suckers, subdivided into two groups, in the dorsal row of the distal part of the hectocotylus and a dorsal lobe complementing the copulatory apparatus. In females of S. boletzkyi sp. nov. the bursa copulatrix is large and, unlike all other species in the genus, has a large fungiform process towards the mantle wall. The discovery of S. boletzkyi sp. nov. increases to 12 the number of NE Atlantic-Mediterranean species of Sepiola.


R e s e a r c h a r t i c l e
urn:lsid:zoobank.org:pub:11B9BCE3-18F9-429F-8EEA-4E232D9E42E0 In the paper describing S. bursadhaesa, the most recently defined Sepiola species, Bello (2013: 498) stated "Because of the […] combination of factors that facilitates speciation [in Sepiola], it is likely that other, still undescribed species of this genus await discov ery."An additional Mediterranean species, viz.Sepiola boletzkyi sp.nov., was discovered in the Aegean Sea and is described in the present paper.

Material and methods
The sepiolines were caught by a hand-operated 1 mm mesh size tulle net, during daytime.In the laboratory they were measured (mantle length) and fixed in 10% formalin in seawater.Afterwards they were preserved in 70% ethyl alcohol.
The sample consists of two specimens which are housed in the Ege Üniversitesi Su Ürünleri Fakültesi Müzesi (ESFM) (Ege University Faculty of Fisheries Museum) of Izmir (sample nos.CEP/1994-1 and CEP/1994-2).Subsequent samplings, carried out in recent times in the same area, did not provide any additional specimens of this new species.
The photographs of anatomical details were taken with a digital camera (DFC 420, Leica, Cambridge, U.K.) attached to a stereo microscope.
Measurements were taken on the preserved specimens, except mantle length, which was also taken before preservation, following Roper & Voss (1983) and Reid (2009).Indices were calculated by expressing each measure as a percentage of mantle length; their acronyms are defined in Table 1 next to each measurement designation.

Diagnosis
Sepiolines with pair of kidney-shaped light-organ lenses on ventral surface of ink sac; suck ers biserial on arms I to III and at least proximally on arms IV; tentacle club suckers in four to eight oblique rows; females with ear-shaped bursa copulatrix on left ventral side of mantle cavity; males with left arm I hectocotylized: suckers and sucker stalks typically arranged in a basal part, a copulatory apparatus comprised of modified suckerless stalks, and a distal part (Bello 2013).

Etymology
From the family name of the eminent teuthologist Sigurd von Boletzky, in recognition of his enormous contribution to teuthology.
Body shape corresponds to general shape of NE Atlantic-Mediterranean Sepiola species (Fig. 2).Mantle sac-shaped, broadly rounded at posterior end, length exceeds width, outline U-shaped.Dorsal mantle margin fused to head by cutaneous nuchal band 27-31% of ML.Ventral mantle margin very slightly sinuate, with very low barely visible rounded projection on each side of funnel (Fig. 2).VML approximately 89-98% of ML.Fins typical for Sepiola, each inserted midway along sides of mantle, broadly rounded; fin length slightly exceeds half ML, fin width about 70-80% of fin length, insertion line about 60% of overall fin length.Head width 65-69% of ML in both specimens.Eyes large, bulging beyond sides of the head, covered by corneal membrane and bordered by secondary eyelid.Funnel long about 60% of ML, covered posteriorly by ventral margin of mantle, funnel free length about 40% of ML; distal end does not reach junction of ventral arms.
Arm formula II = III > IV > I in both specimens.Arms slightly longer in male than in female.Suckers stalked, alternately placed in two series on oral side of all arms, including distal-most part of arms IV.Tentacles thin and delicate.Tentacular club longitudinally lined by protective membranes more developed and ventrally trabeculate (width 1.5-2 mm); distal third of club abruptly tapered, elongate; club suckers equal sized in six oblique rows (Fig. 3).
Overall skin colouration pale violet brown; most chromatophores not expanded.(Actual colouration undoubtedly altered by several year storage in ethyl alcohol.)No particular chromatophore distribution pattern observed.
Mantle-locking apparatus straight, as typical for the genus, long about ¼ ML.
Gladius positioned sagittally on inner face of dorsal mantle, encased in shell sac (see Bizikov 2008 for general description of sepiolid gladius).Gladius reduced, extends from anterior mantle margin to about 45% of ML, posterior tip does not reach posterior end of mantle (as is typical for the genus).Gladius shape similar to those reported for S. bursadhaesa (Bello 2013) and S. aurantiaca (Jatta 1896) (Fig. 4).
Chitinous rachis light tan-coloured, widely open V-shaped in cross section.Gladius bluntly pointed at anterior extremity then gradually enlarges to reach width of about 4% of GL (0.32 mm in examined specimen); posteriorly, two sides of rachis run almost parallel to about 55% of GL and subsequently converge to a point short of posterior end.Point where rachis starts to narrow corresponds to anterior insertion of thin and transparent lateral plates.Posterior part of gladius spear-head shaped where rachis is the shaft and lateral plates the two lateral blades that taper posteriorly to a narrow point exceeding the convergence point of the rachis by about 2.5% of GL.
Beaks indistinguishable from those of other NE Atlantic-Mediterranean Sepiola species (cf.Açik & Salman 2010) (Fig. 5).Female beak measurements are shown in Table 2. Upper beak rostrum uniformly curved; jaw angle obtuse; wing long as anterior lateral wall margin, shoulder (cutting edge) straight, its margin neatly separated at an obtuse angle from rostrum; no grooves at hood sides; no lateral wall  fold; most of lateral wall, posterior part of hood and inferior part of wing transparent.Lower beak comparatively wide; rostrum with blunt tip; jaw edge profile convex, jaw angle indistinct; blunt tooth on shoulder; no notch in hood; lateral wall without fold or ridge, roughly rectangular with lower edge slightly convex, corner faintly produced; wall and wings transparent.

Male
Mature.Spermatophores 6.5 mm long.Mantle cavity does not show any unique traits that allows it to be distinguished from other Sepiola species.Brachial crown dimorphic.Left arm I hectocotylized (see below), arms II bear 6 pairs of enlarged suckers in middle third; arms III not evidently more robust than remaining arms and not bent inward (contrary to several Sepiola species); arms IV bear 3 pairs of enlarged suckers in middle third.
Hectocotylus (left arm I) shorter than right arm I. Basal part (refer to diagram in Fig. 1) bears three equal-sized, not markedly enlarged, suckers, two in ventral and one in dorsal row (the latter missing in holotype as result of damage) (Fig. 6A).Copulatory apparatus formed by fusion of four suckerless stalks, elongated and curved medially, projecting away from arm; ventral-most ones correspond to third and fourth stalks of ventral row; dorsal-most ones derive from second and third stalks of dorsal row, modified into roundish tubercle (Fig. 6A-B).Copulatory apparatus complemented by broad smoothpointed triangular lobe, dorsomedial to tubercle, formed from more than one dorsal suckerless stalks fused with each other (i.e., likely fourth, fifth and sixth stalks of dorsal sucker row); lobe forms groove with tubercle (Fig. 6B-C).
Distal part of hectocotylus somewhat bent, narrow (i.e., two rows of suckers not spread apart to form a spoon-like widening) (Fig. 6A).Arm tip blunt.No groove on hectocotylus lateral side facing right arm I (Fig. 6B).First two suckers of dorsal row (Fig. 6A, suckers 1-2) in distal part smaller than facing suckers of ventral row; followed by six markedly enlarged (Fig. 6A, suckers 3-8), one small (smaller than facing ventral row suckers) (Fig. 6A, sucker 9), two enlarged suckers (Fig. 6A, suckers 10-11).All enlarged suckers about the same size.Distal to enlarged suckers, remaining eight suckers gradually taper to arm tip.Sucker stalks of enlarged suckers proportionally larger and elongated.Suckers of ventral row regularly sized.

Female
Immature.Bursa copulatrix large, roughly ear-shaped, longer than wide; anteriorly extends below left light-organ lens, posteriorly reaches posterior end of mantle cavity (most probably this is not so in mature females with enlarged ovary), medially reaches sagittal mantle cavity septum (Fig. 7A).Folds converge towards centre of bursa where they deepen into a longitudinal opening.Posterior half of bursa bordered by elevated rim; contour elliptical, slightly pointed (like a barge prow).Large fungiform process, towards mantle wall rear of gill insertion, sticking out of bursa laterally from converging folds; stalk of process longitudinally folded; distal end larger than stalk, flat, roughly triangular with rounded corners, might be fused with mantle wall (slight possible scar on it) (Fig. 7B).Abbreviations: a-d = suckerless stalks forming the copulatory apparatus proper (a = ventral-most, d = dorsal-most one, i.e. tubercle); * = lobe in the dorsal row of suckers, 1-11 = sequence of heteromorphous suckers in the dorsal row of distal part. A. Oral view; the arm is slightly tilted, with the tip towards the observer, in order to show details (lobe and small suckers 1-2), otherwise hidden by the four suckerless stalks of the copulatory apparatus.Basal part: only the two ventral suckers are visible, the dorsal one is missing as a result of damage.Copulatory apparatus: the tubercle (d) partially hides the lobe in the dorsal row (*).Distal part: the heteromorphous suckers in the dorsal row are 1-2 small, 3-8 enlarged, 9 small, 10-11 enlarged; the remaining suckers are regular; note that some suckers (2, 3 and 7) are displaced towards the centre of the arm, i.e., not aligned with the others of their same row; it is not known whether this is natural or caused by preservation.B. Inner-lateral view of copulatory apparatus; the lobe (*) forms a groove with the tubercle (d).C. Oblique inner-lateral view of copulatory apparatus; two barely visible longitudinal lines on the lobe (*) are likely fusion lines between adjacent stalks forming it.Scale bars = 1 mm.

European Journal of Taxonomy 144: 1-12 (2015)
Remarks Sepiola boletzkyi sp.nov.can be distinguished from other species in the genus based on the hectocotylus and the bursa copulatrix.As discovered by Naef (1912aNaef ( , b, 1923) ) these copulatory organs are speciesspecific, hence they discriminate the Sepiola species from each other (see also Bello 1995Bello , 2013)).In particular S. boletzkyi sp.nov.belongs to the Sepiola atlantica group as defined by Naef (1923), characterized by the peculiar morphology of the male copulatory apparatus (Fig. 1).
The male S. boletzkyi sp.nov.differs from all other species in the S. atlantica group because it is unique in exhibiting simultaneously the characters (1) homomorphous tip of ventral arms and (2) enlarged suckers of the dorsal row of the hectocotylus distal part that are subdivided into two groups.Character 1 is shared by S. affinis, S. bursadhaesa, S. intermedia, S. robusta and S. rondeletii as opposed to S. atlantica, S. steenstrupiana and S. tridens; character 2 is displayed only by S. atlantica and S. tridens as opposed to all other species.It is not clear to which Sepiola species S. boletzkyi sp.nov. is closer because this new sepioline differs from all four subgroups delineated by Bello (2013) within the atlantica group.Although it shares with S. atlantica + S. tridens the presence of several enlarged suckers of the dorsal row of the hectocotylus distal part (character 2), it neatly differs from them because of the homomorphous tip of ventral arms (character 1).Moreover the grouping of the enlarged suckers in S. boletzkyi sp.nov.diverges from that of the other two species for the number of suckers in both the proximal and midway groups as well as,  3 reports the sequence of enlarged suckers in the three species with two groups of sucker-sizes.
With respect to (S. affinis + S. bursadhaesa + S. intermedia), which have just one group of enlarged suckers in the dorsal row of the distal part of the hectocotylus, S. boletzkyi sp.nov.lacks the groove on the medial side of the same distal part.
Sepiola boletzkyi sp.nov. is easily distinguished from S. rondeletii and S. robusta based on the fact they bear no enlarged suckers on the distal part of the hectocotylus and have 8 rows of suckers on the tentacle club, i.e., 2 more than in S. boletzkyi sp.nov.Lastly S. boletzkyi sp.nov.differs mostly from S. steenstrupiana because of the homomorphous tips of the ventral arms, the structure of the copulatory apparatus, the presence of enlarged suckers on the distal part of the hectocotylus, and the occurrence of 6 rows of suckers instead of 4-5 on the tentacle club.
In addition to the abovementioned differences, the hectocotylus of S. boletzkyi sp.nov. is the only one among the members of the S. atlantica group to have a quite large lobe in the dorsal sucker row next to the copulatory apparatus tubercle.
The female S. boletzkyi sp.nov.may be discriminated from all other Sepiola species of the S. atlantica group by its unique bursa copulatrix.It differs from all members of this group, except S. affinis and S. rondeletii, because of the process projecting towards the mantle wall near the gill insertion; the process in S. boletzkyi sp.nov., however, is fungiform and larger than in both other species.Furthermore the bursa copulatrix of S. boletzkyi sp.nov.extends more, both anteriorly and posteriorly, than that of S. affinis and, in comparison with S. rondeletii, has no caecum (cf. Naef 1923: figs 364 and 368).
The male and female specimens herein described as holotype and paratype, respectively, of S. boletzkyi sp.nov.were assigned to the same specific entity thanks to the similarity with each other in characters other than sexual, primarily the tentacle club, in addition to having been collected in the same place.
In the only available female (the paratype), an immature specimen, the bursa copulatrix reaches the posterior end of the mantle cavity but probably it does not do so in the sexually mature female whose ovary enlarges as oocytes mature.

Discussion
Sepiola boletzkyi sp.nov. is the ninth species of the genus to be discovered in the Mediterranean Sea.It also adds to the six Sepiola species already recorded in the eastern Mediterranean Sea by Salman (2009).
As stressed in the section "Remarks", the new species is assigned, because of its hectocotylus, to the Sepiola atlantica group as defined by Naef (1923), who notably did not grant any genus-group rank to it.We adhere to Naef's concept and do not endorse any generic status for the atlantica group.The species in
2 small, 6 enlarged, 1 small, 2 enlarged the group share, in addition to the general body bauplan, the synapomorphy of the typical hectocotylus and particularly the peculiar copulatory apparatus.According to Naef (1923: 582), in these species "the base of the hectocotylus bears 3 normal suckers, followed by a typical copulatory apparatus which develops from the stalks of 4 suckers, the 2nd and 3rd sucker of the median [i.e.dorsal] row and the 3rd and 4th of the lateral [i.e.ventral] row."These four suckerless stalks are enlarged and fused together to form a typical organ followed by the distal part of the arm that may bear some enlarged suckers in the dorsal row (Figs 1, 6A).At this moment the Sepiola atlantica group includes S. affinis, S. atlantica, S. boletzkyi sp.nov., S. bursadhaesa, S. intermedia, S. robusta, S. rondeletii, S. steenstrupiana and S. tridens.
It is remarkable that three new Sepiola species, all belonging to the atlantica group -namely S. tridens, S. bursadhaesa and S. boletzkyi sp.nov.-have been discovered in the Atlantic-Mediterranean area in the last few years, after a century-long lapse of time since Naef (1912aNaef ( , 1912bNaef ( , 1916Naef ( , 1923) ) described several new Mediterranean sepioline species and, most importantly, disentangled and re-shaped the muddled systematics of the Sepiolinae.
The genus Sepiola displays a great diversity in the Atlantic-Mediterranean area, where it is by far the most speciose cephalopod genus thanks to its 12 species.The reasons underlying this phenomenon were discussed in Bello (2013).The fairly diverse copulatory organs in males (hectocotylus) and, less conspicuously, in females (bursa copulatrix), supposedly give rise to sexual segregation and, therefore, most probably play an important role in sepioline speciation.The discovery of S. boletzkyi sp.nov.corroborates the hypothesis that the NE Atlantic-Mediterranean species of Sepiola are a suite of species that largely arose from in situ speciation on either side of the Strait of Gibraltar after the vicariant event(s) of (recurring) connection to the Atlantic and isolation of the Mediterranean (Bello 2013).
Sepiola boletzkyi sp.nov. is seemingly endemic to the eastern Mediterranean.Its discovery reinforces the statement by Mangold & Boletzky (1988: 326) that "It is interesting that the only cephalopod species exclusively known from the Mediterranean are among the Sepiolinae".Indeed, as shown by Bello (2013), all or almost all Mediterranean species in the genus Sepiola are endemic or quasi-endemic; on the other side of the Strait, the NE Atlantic Sepiola species are also endemic to that area.
The description of Sepiola boletzkyi sp.nov. is based on two specimens only, a male and a female.The collection of additional specimens is necessary to define both the variability of the markedly enlarged suckers in the dorsal row of the hectocotylus distal part, in males, and the shape of the bursa copulatrix in mated females.Moreover, new supplementary material would allow tissue sampling for genetic analyses and DNA barcoding.

Fig. 6 .
Fig. 6.Hectocotylus (left arm I) of Sepiola boletzkyi sp.nov., holotype, ♂, ESFM-CEP/1994-1.Abbreviations: a-d = suckerless stalks forming the copulatory apparatus proper (a = ventral-most, d = dorsal-most one, i.e. tubercle); * = lobe in the dorsal row of suckers, 1-11 = sequence of heteromorphous suckers in the dorsal row of distal part. A. Oral view; the arm is slightly tilted, with the tip towards the observer, in order to show details (lobe and small suckers 1-2), otherwise hidden by the four suckerless stalks of the copulatory apparatus.Basal part: only the two ventral suckers are visible, the dorsal one is missing as a result of damage.Copulatory apparatus: the tubercle (d) partially hides the lobe in the dorsal row (*).Distal part: the heteromorphous suckers in the dorsal row are 1-2 small, 3-8 enlarged, 9 small, 10-11 enlarged; the remaining suckers are regular; note that some suckers (2, 3 and 7) are displaced towards the centre of the arm, i.e., not aligned with the others of their same row; it is not known whether this is natural or caused by preservation.B. Inner-lateral view of copulatory apparatus; the lobe (*) forms a groove with the tubercle (d).C. Oblique inner-lateral view of copulatory apparatus; two barely visible longitudinal lines on the lobe (*) are likely fusion lines between adjacent stalks forming it.Scale bars = 1 mm.

Fig. 7 .
Fig. 7. Bursa copulatrix of Sepiola boletzkyi sp.nov., paratype, ♀, ESFM-CEP/1994-2.A. Ventral view; the dashed line encloses the distal surface of the large fungiform process.B. Oblique ventral view; the fungiform process sticks out of the bursa, laterally to the folds convergence; an arrow points to the longitudinally folded stalk of the process.Scale bar for both figures = 1 mm.

Table 1 .
Measurements and indices of type specimens of Sepiola boletzkyi sp.nov., MLs in square brackets are those taken on fresh specimens prior to preservation.BELLO G. & SALMAN A., Sepiola boletzkyi sp.nov.from the Aegean Sea Brachial crown of mature males displays sexual dimorphic features (see below for detailed description).Arm web reduced; encloses base of tentacles between arms III and IV.