Morphological comparison between Doryteuthis pleii and D. sanpaulensis (Cephalopoda, Myopsida, Loliginidae) from Brazil

The distinction of squid species in the genus Doryteuthis is not easy due to their morphological similarity, lack of conspicuous specific characters, and overlap geographical occurrence. This difficulty has leading to an almost exclusive molecular approach, and a premature neglect of the morpho-anatomy. To emphasize that the squid phenotypic features can be useful to identify, as well as to perform any comparative analyses (such as taxonomy and phylogeny), two close species were selected as outset. Doryteuthis pleii and D. sanpaulensis are common sympatric squids in Brazilian waters, commonly used in fisheries, not so difficult to distinguish by external features of the adult specimens. The samples were analyzed from biometric data to dissections, and the found most expressive characters to distinguish them are the mantle-fin ratio; morphology of the tentacle club, its ratio compared to the mantle length; hectocotylus morphology and nidamental gland morphology. Key-Words. Anatomy; Characters; Squid; Sympatric; Taxonomy.


INTRODUCTION
Loliginidae is a widely distributed squid family of economic importance, extensively targeted as fishery products and with a relevant role in an intricate marine food web (Fields, 1965;Anderson, 2000;Vecchione & Young, 2010a;Granados-Amores et al., 2014). It comprises 47 species distributed in 10 genera, all of which are pelagic (Jereb & Roper, 2010;Vecchione & Young, 2010a;Granados-Amores et al., 2014). Loliginids usually have the posterior end of the fins connected to the mantle and four rows of suckers on the tentacular club [except for Pickfordiateuthis (Voss, 1953) (see Brakoniecki, 1996;Vecchione & Young, 2010a)]. Initially, two of the commonest species in Brazilian coast, Doryteuthis pleii (Blainville, 1823) and D. sanpaulensis (Brakoniecki, 1984), were placed in the genus Loligo, a genus that is now restricted to the Eastern Atlantic (Vecchione et al., 2005).
There is eight species of Doryteuthis, which are sometimes misidentified due to their morphological similarity, the lack of conspicuous, indubitable specific characters to define them, and their overlapped geographical distribution (Vecchione et al., 1998;Vecchione & Young, 2010b).
Nowadays, detailed studies on squid anatomy are scarce, even though morphological data have proved to be very useful in producing phylogenetic hypotheses and defining relationships within mollusk groups (e.g., Simone, 2011;Couto et al., 2015;Dornellas & Simone, 2015). There are more recent papers about molecular analyses than morphological studies. Still, most morphological studies on squids are focused on external characters, barely taking into account the internal anatomy.
Therefore, considering that D. pleii and D. sanpaulensis are relatively locally common, have economic importance in Brazilian waters, and are sympatric, the main purpose of this study is to clarify the differences between these two species, which are commonly misidentified, focusing on both internal and external characters, a more practical approach to determine the species from laboratories to fishmongers. It is not the intention of this paper to perform any statistical analysis, an approach already done elsewhere (Juanicó, 1979), focusing, here, the morpho-anatomical features only. The pioneer study by Juanicó (1979) is commonly used to base the identification of D. pleii and D. sanpaulensis (then called Loligo brasiliensis Blainville, 1823), and has a comprehensive morphometric analysis of the external features and of the gladius.  Distribution: Cape Hatteras (36°N) to northern Argentina (35°S) usually associated with Brazilian warm current. Also including Gulf of Mexico, Caribbean Sea, Bermuda and Bahamian and Caribbean islands (Jereb & Roper, 2010).
Head: Eye diameter approximately 13% of mantle length (Table 2). Width similar to length values.
Chromatophores: Widely distributed, with higher densities on dorsal region including head (around eyes), dorsal midline of mantle (highly concentrated beginning of fins), fins and arms/tentacles. Mature males presenting striped pattern along ventral surface of mantle (Fig. 1A).
Arms: Muscular, broad at base and gradually tapering into thin tips; formula III>IV>II>I (~ lengths: 33 mm, 30 mm, 29 mm, 23 mm); biserial suckers. Hectocotylized portion ~ 39% of left ventral arm length reaching arm's tip (Table 2), only left row of suckers modified into small peduncles. Bar between row of peduncles and row of suckers connecting them in ladder pattern (Fig. 1E).
Tentacle: Club long and thin, ~ 28% ML (Table 2), carpus, manus and dactylus easily defined; 4 rows of suckers diagonally distributed along club, more visible at manus and dactylus regions; 2 central rows at manus region much bigger than other suckers. Each sucker with small chitin ring of teeth inside. Longitudinal muscle around sucker visible (Figs. 1B, 1D).
One central systemic croissant-shaped heart; two smaller peripheral branchial round hearts associated to gills. Two aortae: (i) anterior aorta delivering blood to head, arms and tentacles; (ii) posterior aortic trunk, divided in: (a) visceral aorta, sending blood to posterior part of mantle and visceral organs; (b) mantle aorta, distributing blood to anterior region of mantle. Blood return to heart (i) from posterior vena cava and (ii) from lateral nephridial vessel, both ending in a branchial heart. Branchial artery leaving branchial heart, and ending in gills complex; after this, returning to heart through branchial vein (Fig. 3A).
Digestive system (Figs. 5, 6): Pigmented tip of upper beak short and robust (Fig. 1C). Radula usual for the family; rachidian and first lateral teeth bearing primary large projection and secondary smaller one(s) (Fig. 4A).
Usual digestive system for Loliginidae: buccal bulb occupying ~ half of inner head volume. Esophagus narrow, running straight posteriorly along ~ ⅕ of mantle length. Stomach spherical, wall weakly muscular, occupying ~ 20% of visceral volume, located in middle level of visceral sac; posterior caecum wide, extending up to posterior mantle space. Intestine narrow, originating in YY region of stomach, performing simple loop in anterior region of visceral sac. Anus with two papillae, located in middle level of pallial cavity, preceded by short flexible stalk. Digestive gland wide and located between four retractile muscles of head and funnel. Esophagus passing through digestive gland ventro-dorsally.
Head: Eye diameter approximately 12% ML (Table 4). Length and width very similar to each other.
Chromatophores: Distributed throughout body, especially on dorsal region including head (around eyes); dorsal midline of mantle forming an expressive solid line in region free from fins; few chromatophores clustering in middle of fins and at arms/tentacles ( Fig. 2A). Mature males do not present stripped chromatophores along ventral portion of mantle.
Arms: Muscular, thick at base and distally tapering into slender tips; formula III>IV>II>I (~ lengths: 41 mm, 38 mm, 36 mm, 28 mm); two rows of suckers. Hectocotylus consists of ~ 40% of left ventral arm length extending to arm tip (Table 4). Left row of suckers modified into a simple peduncle whereas right row remaining as suckers. No connection between modified row and suckers, forming valley pattern throughout hectocotylus (Fig. 2E).
Tentacle: Club long and broad, ~ 37% ML (Table 4). Carpus, manus and dactylus uniformly distributed, with boundaries not very sharp, hindering their definition (especially between manus and dactylus); 4 diagonal rows of suckers along club as stated for D. pleii; 2 central rows at manus not so different in size from other suckers (Fig. 2B). Each sucker with small ring of teeth apparently at surface of sucker's aperture. Longitudinal muscle around suckers present (Fig. 2D).
Circulatory and central nervous system: Similar to preceding species. No apparent morphological differences (Fig. 3A).
Digestive system: Pigmented tip of upper beak is long and slender (Fig. 2C). Remaining structures similar to preceding species.
Male genital system: Similar to preceding species. No apparent morphological differences (Figs. 6A, 6B) except for hectocotylus above described.
Female genital system: Similar to preceding species except for nidamental glands with prominent thin tip (Fig. 5D).

DISCUSSION
Doryteuthis pleii and D. sanpaulensis are really similar to each other, sometimes difficult to separate in the field and in preserved samples. The main differences between them resides in the gladial features and in external proportions (Juanicó, 1979;Brakoniecki, 1984). However, the overall anatomy of both species described herein showed additional distinguishing characters. The tentacular club of D. pleii has two central rows of suckers larger than the marginal ones (Fig. 1B), whereas D. sanpaulensis has four similarly sized rows of suckers (Fig. 2B), where the marginal rows are just slightly smaller than the central ones.  Table 4. Proportions between characters of Doryteuthis sanpaulensis. FL-ML: Proportion between fin length and mantle length (%); ED-ML: Proportion between eye diameter and mantle length (%); HtL-Lft AIV: Proportion between hectocotylized portion and left ventral arm length (%). CL-ML: Proportion between tentacular club length and mantle length (%).
The mantle itself does not have large differences between these species. However, the mantle-fin ratio in D. pleii usually reaches approximately 40-50%, hardly no more than ½ of mantle length (Fig. 1A), while in D. sanpaulensis this ratio usually stays around 60% or more ( Fig. 2A). Additionally, Brakoniecki (1984) stated that the mantle-fin ratio of D. pleii are more similar to D. gahi than to D. sanpaulensis.  Cohen (1976) stated that the hectocotylized portion of D. pleii is no more than ½ of the left ventral arm length, which was confirmed here. The hectocotylized portion is practically the same for these two specimens, comprising 39-40% of the left ventral arm length. However, the morphology of the hectocotylus differs between them. Doryteuthis pleii shows a bar connecting the modified and unmodified row, forming a ladder pattern (Fig. 1E), whilst D. sanpaulensis does not have any kind of structure connecting the rows, forming a valley between the rows (Fig. 2E).
The eye diameter of D. pleii has been referred as about 14-19% of mantle length (Brakoniecki, 1984); however, this study has obtained the eye diameter of around 13%. This difference has been interpreted as a variation in the southern population. As evidenced herein, the eye diameter in D. sanpaulensis is around 12% ML.
The statistical morphometric analyses done by Juanicó (1979) already showed the distinction between both species, including ontogenetic and dimorphism variation. The preliminary results of the data of the present paper greatly coincided with those of Juanicó (1979), which was also partially included in Perez et al. (2002), dispensing the need of additional analyses. This paper remains, then, focused on the visible morpho-anatomical features of adult specimens.
The internal anatomy does not show significant differences between these two species. The circulatory and the nervous systems have practically the same features in the two species (Fig. 3). Regarding the digestive system, only the beak structure has shown some differences. The pigmented tip of the upper beak of D. pleii is short and robust (Fig. 1C), whereas that of D. sanpaulensis has a long and slender tip (Fig. 2C). Moreover, the radulae also did not have expressive differences to distinguish the species (Fig. 4). Doryteuthis pleii and D. sanpaulensis have typical squid radulae: rachidian and first lateral teeth with a primary large projection and secondary smaller one(s) (Fig. 4).
As for the genital system, the nidamental glands are more uniform in D. pleii females, without a slender tip (Fig. 5B), whilst in D. sanpaulensis females, the nidamental glands have a more prominent, thin anterior tip (Fig. 5D). On the other hand, there were no significant differences between the male genital system of both species (Fig. 6). In the case of the male genital system, spermatophores are good sources of distinguishing characters, since they are highly complex and a key structure to differentiate species (Marian & Domaneschi, 2012;Marian, 2012).
Even though there are interesting characters listed in this study that can be used to further distinguish D. pleii from D. sanpaulensis, there are also some characteristics that might bring them phylogenetically closer. This is the case of the size of hatchlings and eggs (Barón, 2003b); the slightly exclusive internal anatomy; and the morphology of the hectocotylus [Brakoniecki (1986) once clustered D. pleii and D. sanpaulensis together using this character]. The key to the subgenus Doryteuthis, as proposed by Vecchione et al. (2005), is based on the hectocotylized portion extending to the arm tip, and on the thickened gladius vane for Doryteuthis; a characters absent in Amerigo; for D. sanpaulensis, the hectocotylized portion is similar to those in the Doryteuthis s.s. Therefore, the undescribed subgenus of D. sanpaulensis might be phylogenetically closer to Doryteuthis than to Amerigo. Certainly, more studies are required to clarify this relation among Doryteuthis species as well as a complete study to describe the subgenus of D. sanpaulensis, or to consider it definitively as Doryteuthis.
As the separation between Loligo and Doryteuthis has been essentially based on molecular results, another intention of this paper is furnishing additional, morphological subsides for genus' definition. Possibly further studies on more species of these two taxa in the same level as described here can supply identification by simply inspection, dispensing laboratorial procedures or morphometric analyses. On the other hand, it is recognized that the relative uniformity of the internal anatomy of both species herein studied cannot resist to further investigation, such as more details of the digestive system (e.g., odontophore and beak muscles), of the brain, etc. These details are still being developed, and are not the present scope.

CONCLUSIONS
1) Doryteuthis pleii and D. sanpaulensis are really close taxa, and possibly belong to the same subgenus (Doryteuthis s.s.). 2) Despite their similarities, at least details and proportions of the mantle color and form, mantle-fin ratio, tentacular club, hectocotylus, gladius, and eye, can provide relative easy identification between there both sympatric species. 3) The internal anatomy, in the present level of details, is relatively uniform between both species; significant differences are fond in beak and nidamental glands.