Morphological and Morphometric Variability of the Squid Lolliguncula Brevis (mollusca: Cephalopoda) in Brazilian Waters: Evidence for Two Species in the Western Atlantic?

Morphological and morphometric variability of the squid Lolliguncula brevis (Mollusca: Cephalopoda) in Brazilian waters: evidence for two species in the western atlantic? ABSTRACT Morphological and morphometric variability of the small-sized coastal squid Lolliguncula brevis was assessed along the largest part of its latitudinal range in the southern hemisphere, off the Brazilian coast (8° S-27° S). A general homogeneity in form was found throughout the entire latitudinal range. In terms of body proportions, no latitudinal gradients were evident, but a few local " morphotypes " could be distinguished. The distinctive egg mass morphology, and size and form features, including a hectocotylus 20-40% longer than the opposing ventral arm and the presence of suckers on the buccal membrane, indicated that Lolliguncula from northern and southern hemispheres might comprise two different taxa.


INTRODUCTION
Lolliguncula Steenstrup, 1881 is one of the ten genera included in the Family Loliginidae (Vecchione et al. 2005), which comprises the most abundant squid forms found in coastal areas and continental shelves worldwide.Recent taxonomic reviews have established the validity of four to five Lolliguncula species, all of them small-sized squid occurring in warm coastal habitats such as shallow bays and estuaries (Vecchione et al. 1998(Vecchione et al. , 2005)).The geographic distribution of the genus is centered in the Panamá region, as most species are limited to tropical and subtropical waters off both North and South American shores.L. panamensis, L. argus and L. diomedeae occur in the Pacific Ocean, whereas L. brevis occurs in the Atlantic side of the Americas and the Caribbean.
Originally included in the genus Loligo, Lolliguncula brevis was firstly described by Blainville in 1823 from specimens collected off South America.Solely based on male hectocotylus morphology as a diagnostic feature, this description was reviewed and complemented from the 1910´s onwards as other morphological characters exhibited by North Atlantic specimens were compiled (Berry 1911, 1913, Voss 1956, Abbott 1974, Roper 1978, Roper Anais da Academia Brasileira de Ciências ( 2012) 84(4): 1015-1028 (Annals of the Brazilian Academy of Sciences) Printed version ISSN 0001-3765 / Online version ISSN 1678-2690 www.scielo.br/aabcTÂNIA ZALESKI, JOSÉ A. ANGEL PEREZ and ANA LUIZA GANDARA-MARTINS et al. 1984).The species was then considered a single taxum with a wide latitudinal distribution in the Western Atlantic, occurring from 40° N (South Carolina) to 27° S (Santa Catarina) including the Gulf of Mexico and the Caribbean (Roper et al. 1984).
Records of the species in Brazilian waters only reappeared in the late 70´s review of Brazilian cephalopod fauna by F.J. Palacio (unpublished data), and subsequent reports of scientific surveys off the Brazilian coast (M.Juanicó, unpublished data; Haimovici and Andriguetto-Filho 1986).Those records were compiled in more comprehensive reviews of cephalopod Brazilian fauna (Rios 1985, Haimovici et al. 1994, Perez and Haimovici 1991, Haimovici and Perez 1991), which extended the species distribution range in southern Atlantic as far south as Santa Catarina state (28° S).
Morphologic and morphometric variability of Lolliguncula brevis was preliminarily analyzed by M. Juanicó (unpublished data), who noticed possible geographic patterns along the Brazilian coast.Later, a detailed study conducted by Simone (1997) evidenced differences between Brazilian and North American forms, suggesting that more than one species could have been included under the same name, and that a general review of this taxum was required.The present work explores both M. Juanicó's (unpublished data) and Simone's (1997) hypotheses providing a detailed morphometric analysis of L. brevis collected along a significant part of its latitudinal range in the southern hemisphere, off the Brazilian coast (8° S -27° S), complemented with a well development morphological description.

MATERIALS AND METHODS
This study was based on a total of 196 specimens of Lolliguncula brevis collected in Brazilian waters, including 112 females and 84 males (Table I).These specimens were obtained from 11 localities along the Brazilian coast, from Pernambuco state (8° S) to Santa Catarina state (27° S) (Figure 1).The specimens of Rio de Janeiro state were part of the mollusca collection of Museu Nacional da Universidade Federal do Rio de Janeiro (MNRJ) (Lots 5,635;5,634;5,638 and 5,636).The remainders were collected in situ mostly by trawling in coastal areas.Egg masses were obtained from shrimp trawls over shallow sandy/muddy bottoms in Balneário Camboriú bay (Santa Catarina state; n = 17 egg masses) and Guaratuba bay (Paraná state; n = 4 egg masses).All material was fixed in formalin 4% and preserved in ethanol 70%.Specimens were examined in detail for the general external morphology, and measurements were based in Roper and Voss (1983): mantle length (ML), mantle width (MW), length of siphon (SL), fin length (FL), fin width (FW), head length (HL), head width (HW), eye diameter (ED), intra-orbital distance (IOD), length of the right arms (ALI, ALII, ALIII, ALIV), width of the right arms (AWI, AWII, AWIII, AWIV), right tentacle length (TL), tentacular club length (TCL), sucker diameter (SD), number of suckers in arms (SNI, SNII, SNIII, SNIV), total number of suckers in the hectocotylus (SNHe), number of suckers in the tentacle (SNT), number of right gill lamellae (GLN).In males, the length and width of the hectocotylus were taken (HeL and HeW respectively) as well as length and width of the opposing arm.The gladius was dissected from the gladius sac and measured for the total length (GTL), vane width (GVW), rachis length (GRL) and rachis width (GRW).Individual eggs and egg capsules were measured for the total length, the number of eggs per capsule and the number of capsules per egg mass was also recorded.
The radula of additional ten females and eight males caught off Santa Catarina state coast was extracted from the buccal mass and prepared for observation under JEOL JSM 6360LZ, a Scanning Electron Microscope.I. TÂNIA ZALESKI, JOSÉ A. ANGEL PEREZ and ANA LUIZA GANDARA-MARTINS All measurements were log-transformed and their variability tested for the effect of sex and collection locality using an Analysis of Covariance (ANCOVA), where each measurement was the dependent variable, sex or locality were effect factors, and mantle length (ML) the covariate.Each ANCOVA was preceded by a test for homogeneity of slopes (Zar 1982) where the interactions between the effect factors and the covariate were tested.When such interactions were significant the ANCOVA was not applied.
A Principal Component Analysis (PCA) was applied as a variable reduction technique, and aimed at assessing morphological similarities among the examined specimens and the characterization of eventual morphotypes (Hair-Júnior et al. 1995, Monteiro andReis 1999).PCA was applied for males and females separately and included the following measurements ML, MW, FL, FW, HL, HW, IOD, ED, AL I, AL II, AL III, AL IV, AW I, AW II, AW III, AW IV.These variables were chosen because they were available in most examined specimens, thus allowing the analysis to include the largest sample size.In males, hecocotylus length (HeL) and width (HeW) were also included.All variables were standardized to preserve allometric relationships, variance and equalize errors existent in wide size variations (Bookstein et al. 1985, Voight 1991), using the formula: at the posterior end (Figure 2A, B and C).The dorsal margin was wide with a distinct dorsal lappet marking the anterior end of the gladius.Ventrally, the mantle margin was slightly concave at the basis of the siphon.This concavity was delimited by a pair of lappets that marked externally the position of mantle locking apparatus.Mantle width at its midpoint varied between 26% and 44% of the ML.Fins were round-shaped (Figure 2A, B), wider at their midportion (78% of FL), their length reaching 35-45% of the length of the mantle.
Head was as wide as the mantle or slightly narrower than it (Figure 2A, B).Head length reached, on average ¼ of ML.Eyes were mediumsized, reaching approximately 68% of head length.Siphon was compact, extending anteriorly to the approximate midpoint of the eyes (Figure 2C).The funnel locking cartilages had a straight, groove and the mantle-locking cartilages were straight ridges.
Arms were medium-sized reaching 25-75% of ML.Most frequent arm formulae was IV≈III>II>I.All arms presented poorly developed sucker protecting membranes that extended along the margin of part of its length.Arm III, however, was bordered by a well-developed swimming membrane extending along its entire length.A total of 31-103 suckers, disposed in two rows, were counted on the oral surface of each arm, on average.Sucker chitinous rings had 7-8 squared similar teeth on half of the ring perimeter, being the other half smooth.The seven-lobbed buccal membrane contained a variable number of the small suckers on the oral surface.
Males' left ventral arm was modified into a hectocotylus, reaching 60% of mantle, being noticeably longer than the opposing ventral arm (Figure 2B; 5B).Suckers at approximately 1/3 of the distal dorsal row were gradually reduced (the 1 to 3 proximal modified suckers being greatly reduced in diameter) toward the arm tip.These modified suckers lie on the tip of long triangular pedicels, which diminish in size distally (Figure 2E).Four males caught at "Baía Sul" (the Where SV i is the i-eth standardized variable, V i is the i-eth original variable, V m is the variable mean and σ the variable standard deviation.

RESULTS
All examined specimens were included in the genus Lolliguncula based on rounded fins and a spermatophore-pad near the left gill (Brakoniecki 1980).Mantle was short, cylindrical and slightly rounded southernmost locality sampled) had a distinctively bare hectocotylized portion of the left ventral arm, without suckers or pedicels.
Tentacles were long and laterally compressed.Clubs were bordered by a short swimming membrane.Suckers were larger at the manus and smaller at the dactylus decreasing in diameter towards the distal end of the club.These suckers were arranged in approximately 27 transversal and four longitudinal rows.Chitinous rings of club suckers had pointy teeth (Figure 2G).
Externally, preserved specimens were normally white to pale yellow.Dark chromatophores were scattered over the entire mantle surface, being more concentrated on the dorsal surface and parti cularly along the external gladius outline.Some specimens presented a darker band on the ventral longitudinal midline (Figure 2C).Chromatophores also covered both dorsal and ventral surfaces of the head.The siphon was only pigmented on the ventral surface.Arms and tentacles were pigmented at their aboral surfaces.
Beaks were light in color and moderately short (Figure 2H and I).Radula was composed by chitinous teeth regularly arranged in seven longitudinal rows.Rachidian (central) teeth were tricuspid, the mesocone being longer and pointier than the lateral cuspids.The first lateral teeth were longer than the rachidian teeth and bear two cuspids, the central cuspid being was than the marginal one towards.The second lateral teeth were long and formed by a single triangular cuspid.The third lateral teeth were slightly long with round borders, rectangular marginal plates were present (Figure 3).
The gladius was translucid, moderately short with a wide vane (vane width reached approximately 20% of gladius length).Rachis was narrow (mean width 1.6 mm) with a pointed anterior end (Figure 4).The anterior margin of the vane was straight and continuous to the rachis margin.Posteriorly the vane was round-shaped.Its margins were slightly curved and not thickened laterally.Internally, paired gills bear 70 lamellae each one.
In mature females, the genitalia (ovary, oviduct, nidamental glands and oviducal gland) occupied approximately ⅔ of the mantle visceral space.The oviduct was single and ran parallel MORPHOLOGICAL AND MORPHOMETRIC VARIABILITY OF L. brevis to the digestive caecum.Developed nidamental glands were lamellar and bulky, occupying most of the visceral cavity.The oviducal gland was also greatly developed in mature females and located on the left side of the mantle.Mated females had spermatangia inserted in the muscles of the inner mantle, at the basis of the left gill in the vicinity of the oviduct aperture (Figure 5C, D).In mature males the testis was a white and conical mass located at the posterior end of the visceral cavity.The terminal organ or penis was wide and with a thin and translucid wall.Spermatophores were long and thin.The cement body was long and slender, occupying approximately 1/3 of the spermatophores total length.
Egg masses from both "Guaratuba" and "Balneário Camboriú" were similar in form.These were clusters/clutches of 18-73 short and globuleshaped gelatinous capsules, each one with a peduncule whose length never exceeded half of the capsule total length (Figure 5E, F).These capsules were fused by their peduncles into a common stalk by which the cluster was attached to the substrate.When immersed in water egg clusters formed a sphere with 6-7 cm radius.
Egg capsules were 9.4-28.5 mm long.Each capsule was formed by a string of eggs enclosed in two gelatinous layers, an inner layer that held the eggs together in a spiral thread, and an outer one that enveloped this thread and delineated the capsule and also the peduncule.Each capsule enclosed 1-38 eggs/embryos (mean 18 ± 0.4 SEM, n = 391).Developmental stages observed were 7-27 (according to Arnold 1971 andHunter andSimon 1975).The chorion was ovate (with 1.3-1.7 mm length in the longest axis) in the youngest stage observed.The length increase to 2.5-3.5 mm during organogenesis (stages 18, 19, 20, 21) (Figure 6).

MORPHOMETRIC ANALYSIS
Males and females differed in all but one body measurement (SNT, Table II).This justified the following analysis (ANCOVA) to be conducted for each sex separately.In both sexes, almost all body measurements varied significantly among localities (Table III).
The first and second rotated axes (Factors I and II) generated by the PCA applied to female body measurements accounted for 54% and 15% of total variance, respectively.Higher scores of the first axis were attributed to females with a longer mantle length, longer arms and longer and wider fins.In the second axis, high scores were determined by more robust (wider) arms (Table IV).When examined females were plotted according to the scores determined by factors I and II.No distinct groups could be spatially observed (Figure 7A).
In males, the first and second PCA rotated axis (factors) accounted for 43% and 12% of the total variance, respectively.Specimens scored by factor I were positively influenced by the length of their head, arms and mantle, whereas factor II scores were mostly driven by the positive effect of hectocotylus length and negative effect of arm IV length, fin length and mantle length (Table IV).The spatial representation of examined males, as scored by factors I and II, allowed the distinction of a few regional groups or "morphotypes" (Figure 7B).The most distinctive ones originated from (a) "Cananéia", generally smaller, with narrow head, short to medium arms, short fins, and long hectocotylus; (b) "Camamu", slightly larger and with arms longer than the ones in the group from TÂNIA ZALESKI, JOSÉ A. ANGEL PEREZ and ANA LUIZA GANDARA-MARTINS Cananéia; and (c) "Baía Sul", with wide head, long arms and short hectocotylus.Squids from localities separated by long distances such as "Camamu" and "Guaratuba" were relatively similar.On the other hand; squids from relatively close localities such as "Baía Sul", "Penha" and "Cananéia" appeared as distinct forms in the bidimensional space.

DISCUSSION
Lollingunculla specimens collected off Brazilian coast between 8°S -27° S were morphologically similar and probably comprise a single taxum.Males morphometric analysis, however, revealed geographic dissimilarities in body proportions and the differentiation of local "morphotypes" within the sampled geographic range.Such dissimilarities could not a priori be associated with the distance between localities or latitudinal gradients; squid groups of neighboring localities could be more dissimilar than groups nearly 2,000 km apart.That, in turn, suggests that the population structure of L. brevis along its wide latitudinal range can be complex, and exclusive phenotypes can be maintained in relatively small and enclosed areas, such as bays and estuaries.Two main hypotheses can be drawn as plausible causes of such variability in form.
First, local forms could be the outcome of character plasticity as influenced by local environmental (temperature, salinity, food availability) regimes.Notwithstanding being influenced by the overall latitudinal climate gradient, most localities are associated to complex estuarine systems (i.e."Camamu", "Cananéia", "Guaratuba", "Pontal do Sul", "Rio das Ostras"), each one of them with very particular environmental conditions (Castro-Filho et al. 1987, Pires-Vanin and Matsuura 1993, Panitz 1994).Phenotypic plasticity is well documented in cephalopods, including some extreme cases such as Loligo forbesii, in which forms inhabiting waters of the vicinity of the Azores Archipelago differed radically in size and form from more typical forms inhabiting the European shelf (Brierley et al. 1993).
A second possibility could be the intraspecific phenotypic variation as determined by a limited gene flow among local groups, leading to some degree of genetic isolation.In cephalopods, as in most marine invertebrate and fish groups, such limitation could be associated to (a) the dispersion capacity of planktonic initial life stages, and (b) to the locomotion power of nektonic adults.In the first case, the smaller the hatchlings, the longer the duration of the planktonic life and higher is the dispersion capacity (Boletzky 1987, Vecchione 1987).Lolliguncula are generally large-egged, with a relatively short nektobenthic planktonic life in comparison with Loligo species, for example (Hall 1970, Vecchione 1982, Boyle and Rodhouse 2005).That feature per se suggests limited paralarval dispersion.
Additionally, as adults, L. brevis may not be able to perform long displacements owing to the elevated locomotion demands characteristic of small-sized squid.In fact, as squid body increases, velocity is also increased while transportation costs decrease (O'Dor 1988).Loligo opalescens, which reach sizes as large as 13 cm ML, have its maximum migratory amplitude estimated at around 600 km.Although this species is distributed along nearly 3,500 km off the west coast of North America, three morphologically distinct populations are found in three adjacent  Females and males mean body measurements (± SEM) and the results of statistical tests are indicated.Each ANCOVA (Analysis of Covariance); was preceded by a test for homogeneity of slopes (THS).The asterisk indicates significant differences between sexes (α=0.05).
zones, suggesting that the migration amplitudes could be much smaller (Kashiwada and Recksiek 1978).L. brevis is, on average, much smaller than L. opalescens, thus their migratory capacity is expected to be even more restricted.Such limited migratory amplitude, combined with the effect of local environmental conditions, could result in slightly distinct forms of Lolliguncula brevis along Brazilian large and diverse coastline.
In the North Atlantic and Caribbean waters, the validity of a single taxum, L. brevis, has been generally supported (Jereb et al. 2010).Nevertheless, the present analysis, including a large number of specimens collected along a significant portion of the species distribution in the South Atlantic, reinforces, in accordance with a previous study (Simone 1997) that northern and southern hemisphere forms cannot be classified under the same name.Southern males and females reached body sizes as large as 49.0 and 69.5 mm ML, respectively, being consistently smaller than Lolliguncula brevis recorded off Central and North America (Table V).In contrast with northern forms (Roper et al. 1984), Lolliguncula brevis collected off Brazil are also less robust, with a slender cylindrical mantle (a feature also reported by Simone 1997) and exhibit shorter and narrower fins.Additionally, the morphology of egg capsules and egg masses collected off southern Brazil greatly differs from the observed in the North American coast.Hall's (1970) description of Loliguncula brevis spawning in Tampa Bay, Gulf of Mexico, refers to long egg-capsules fixed individually to the substrate, enclosing 56-88 eggs, whereas the ones collected off southern Brazil were five times shorter, united by a common stalk and fixed as a bunch, enclosing 1-38 eggs (Table V).
Southern L. brevis males were also distinguished from northern forms by exhibiting a hectocotylized arm bearing 17 modified suckers and 20-40% longer than the opposing ventral arm.Roper et al. (1984) reported these arms as subequal in north Atlantic Lolliguncula.Lastly, unlike the specimens collected off Brazil, northern Loligunculla do not seem to bear suckers on the buccal membrane (Roper 1978), although some uncertainty exists about this character (Roper 1978).
Morphological differences between northern and southern forms, as evidenced in this study,  1956), although this information is not completely clear in Blainville's original description and could be as far north as the Caribbean.In any case, a major redescription of both forms will be necessary, and possibly a revalidation of some of L. brevis at least four available synonyms (Vecchione et al. 1998).

Figure 1 -
Figure 1 -Study area and positions where Lolliguncula brevis were sampled for morphological and morphometric analysis.Three sectors of the Brazilian coast are presented in detail depicting specific localities and coastline features.Number of females and males collected per sample, locality and latitude-longitude points are indicated TableI.

Figure 4 -
Figure 4 -Dorsal and lateral aspects of gladius of Lolliguncula brevis.

Figure 5 -
Figure 5 -Anatomical features of Lolliguncula brevis of Brazil.A-Suckers on second arm.B-Hectocotylus of male (white arrow: normal sucker and black arrow: modified sucker).C-and D-Spermatangias inserted in the inner mantle wall of a female.E-Egg mass, i.e., egg capsules fused by their peduncles into a common stalk.F-Detail of egg an capsule.

Figure 6 -
Figure 6 -Increase the size of the chorion of the L. brevis in relation to the stages of egg development.

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MORPHOLOGICAL AND MORPHOMETRIC VARIABILITY OF L. brevis

TABLE II Summary of morphometric analysis of Lolliguncula brevis collected off Brazilian coast.
TÂNIA ZALESKI, JOSÉ A. ANGEL PEREZ and ANA LUIZA GANDARA-MARTINS