(BRACHYURA: MAJOIDEA: PISIDAE) DESCRIBED FROM LABORATORY REARED MATERIAL AND A REAPPRAISAL OF THE CHARACTERS OF PISIDAE

The complete larval stages of Notolopas brasiliensis are described from laboratory reared material, with emphasis on the external morphological features of Majoidea, and compare the morphology of N. brasiliensis with other genera of Pisidae. Larval development of N. brasiliensis consists of two zoeal stages and one megalopa. The duration mean of each zoeal stage was 4.2 ± 1.0 days for Zoea I and 3.8 ± 0.7 days for Zoea II, the megalopa instar appearing 8.1 ± 0.4 days after hatching. The characters previously used to define larval forms of Pisidae are either symplesiomorphic or potentially highly homoplastic. As well, was observed that there are no common sets of larval characters that would define Pisidae nowadays. However, was showed that only a combination of characters could differentiate Notolopas from other pisid genera.


INTRODUCTION
The understanding of evolutionary relationships amongst crustaceans is largely based on adult morphology, and larvae remain a much neglected source of characters that may help solve relationships among taxa.In few particular cases, larval characters have been shown to be useful in phylogenetic inferences (Clark & Webber, 1991;Baisre, 1994;Marques & Pohle, 1995, 1998, 2003;Pohle & Marques, 1998, 2000;Maas & Waloszek, 2001).However, we are still at the stage of providing well detailed descriptions of larval forms that would allow us to make phylogenetic inferences based on those characters for many higher taxa within Brachyura.
The Majoidea (sensu Martin & Davis, 2001) is one of the most diverse groups within Brachyura, with ap-proximately 900 species worldwide (Provenzano & Brownell, 1977).In the Southwest Atlantic this group is represented by approximately 80 species in 45 genera arranged into eight families, in which the family Pisidae is represented by 11 genera with 14 species (Melo, 1996).
The spider crab Notolopas brasiliensis Miers, 1886 is known to inhabit sandy, muddy or gravel bottoms from the intertidal region to depths of 30 m on the coasts of the western Atlantic of Colombia, Venezuela and Brazil.In Brazilian waters, this species is distributed from Amapá to São Paulo States (Melo, 1996).The purpose of this study is to describe the complete larval stages of Notolopas brasiliensis, reared under laboratory conditions, with emphasis on the external morphological features of Majoidea, and compare the morphology of N. brasiliensis with other genera of Pisidae.

MATERIALS AND METHODS
Two ovigerous specimens of Notolopas brasiliensis were collected in March 1998 in Ubatuba, São Paulo, Brazil (23°26'18"S, 45°02'30"W) by trawling in depths of 10 m.The specimens were held in an aquarium until hatching, which occurred at night for both females.After hatching, 50 of the most active, positively phototactic larvae from each female were reared individually in 70 ml acrylic jars containing 30 ml of filtered seawater.The remaining larvae were kept in mass culture as extra specimens to be used for morphological description.
Newly hatched larvae were fed ad libitum with Artemia nauplii.Sea water was changed, and specimens were inspected and fed daily.All acrylic jars were washed in fresh water and air-dried before re-use with fresh seawater in the following day.Mean daily water temperature in the tank was 24° ± 1°C.Average salinity was 32.A 14L:10D photoperiod was maintained.
Whenever possible, a minimum of five specimens of each stage, from each females, were dissected for morphological description, and intra-specific observation.For slide preparations polyvinyl lactophenol mounting medium was used with Acid Fuchsin and/or chlorazol black stains.
The description of setae follows Pohle & Telford (1981), but here includes only analysis by light microscopy (LM), using an Olympus BH-2 microscope with Nomarski Differential Interference Contrast and drawing tube.Some of the setae designated as plumose herein may be plumodenticulate setae due to the lower resolution limits of LM as compared to scanning elec-tron microscopy (SEM).Description guidelines of Clark et al. (1998)

RESULTS
Larval development and description -Larval development of Notolopas brasiliensis consists of two zoeal stages and one megalopa.The duration mean of each zoeal stage was 4.2 ± 1.0 days for Zoea I and 3.8 ± 0.7 days for Zoea II, the megalopa instar appearing 8.1 ± 0.4 days after hatching.Only morphological changes are described for the second zoeal stage.

Description
Notolopas brasiliensis Miers, 1886 First zoea (Figure 1) Carapace (Figure 1A) -Dorsal spine curved, and short straight rostral spine not extending beyond antennule; lateral spines absent.On ventral margin with densely plumose "anterior seta" posterior to scaphognathite notch, followed by 5 additional sparsely plumose setae.Eyes sessile.Frontal area between dorsal and rostral spine forming a distinct swelling with strong muscle bands and bearing small protuberance with dorsal organ (sensu Martin & Laverack, 1992).Additional small knob with dorsal organ posterior to dorsal spine.One pair of simple or sparsely plumose setae present posterior to the dorsal spine.
Telson (Figure 1J) -Bifurcated, distinct median notch, 3 pairs of serrulate setae on inner margin; each furcal shaft proximally bearing minute lateral spine, furcal shafts and spines covered with rows of spinules to just below tips.
Second zoea (Figure 2) Carapace (Figure 2A) -Eyes stalked.Three additional pairs of simple or sparsely plumose setae, two pairs just above eyes, another at base of dorsal spine.Lateral margin anteriorly to posteriorly now with 2 densely plumose and 6 plumose or plumodenticulate setae.

Megalopa (Figures 3 and 4)
Carapace (Figure 3A) -Longer than wide, narrowing anteriorly, with small rostrum deflected slightly ventrally; lateral and dorsolateral ridge extending from eyes to the beginning of branchial area, two additional pairs of dorsal protuberances near border of gastric area.Two small protuberances on the urogastric region, and a pair of tubercles on the metabranquial region.Surface with mostly simple setae as shown.
Antennule (Figure 3B) -Three-segmented peduncle with two simple setae on middle and single seta on distal segment; unsegmented endopod with one subterminal plumodenticulate and 2 terminal simple setae; threesegmented exopod with naked proximal segment, single plumodenticulate seta and 7 aesthetascs on middle segment, and distal segment with 4 aesthetascs with aesthetasc-like apical seta.
Telson (Figure 4G) -Rounded posteriorly, bearing a pair of dorsal setae.Some specimens bear variously reduced setae on the posterior margin.

DISCUSSION
This study reports for the first time larval stages from Notolopas.Previous accounts of the larval features within Pisidae have addressed the larval stages of 30 species within 15 genera (Table 1).However, some authors have argued that there are no larval characters that would define this family (e.g., Pohle & Marques, 2000;Marques & Pohle, 2003).Ingle (1979) discussed character sets for families within Majoidea, and hence postulated some larval features that would characterize pisid larvae.His character set for this family included: absence of carapace lateral spines, rostral and dorsal spines of moderate length, one spine on the telson fork, dorsolateral processes on the 2 nd abdominal somite, rarely on the 3 rd ; posterolateral processes on the 3 rd -5 th abdominal somite often short; basis of the 2 nd maxilliped with not more than 3 setae; antennal exopod with subterminal setae.However, as we have accumulated larval descriptions over the years, these characters became inadequate to represent pisid species with known larval development.
A detailed exam of the characters used by Ingle (1979) to define larval forms of Pisidae shows that most of these characters are either symplesiomorphic or potentially highly homoplastic ones based on previous phylogenetic studies on larval morphology (Clark & Webber, 1991;Marques & Pohle, 1998, 2003;Pohle & Marques, 1998, 2000).For instance, the absence of lateral spines on the carapace is also observed in larval forms within Mithracidae, Epialtidae, Inachidae, Inachoididae and some members of Majidae.The presence of dorsolateral process on the 2 nd abdominal somite can be found in some species of Mithracidae, all Majidae, Epialtidae, Inachidae, Inachoididae.The presence of 3 setae on the basis of the 2 nd maxilliped is shared with species within Majidae, Mithracidae, and most Epialtidae, Inachidae and Inachoididae.Finally, presence of subterminal setae on the exopod of the antenna can also be observed in members of Majidae, Mithracidae, Epialtidae and most Inachoididae (Marques & Pohle, 1998, 2003;Pohle & Marques, 1998, 2000;Santana et al., 2004).Thus, we suggest, as previous works (e.g., Santana et al., 2004), that these characters should not be used to define the larval forms within Pisidae.In addition, as have been asserted for many families within Majoidea (Pohle & Marques, 2000;Marques & Pohle, 2003), there is no unique larval characters and/or combinations of them that would distinguish larval stages of Pisidae from other members of Majoidea.
In contrast to other families of Majoidea in which larvae are difficult to differentiate because of the great consistency of larval morphology within it (e.g., Mithracidae) -especially in zoeal stages (Santana et al., 2003), larvae of Pisidae are difficult to identify because of the great heterogeneity of its larval forms (Santana et al. 2004) showing characters resembling other families.The corollary is the difficulty to find common sets of characters that would define the group as mentioned above.Be that as it may, Santana et al. (2004) compared larval morphology within Pisidae and pointed out some characters that could suggest Pisidae as a phenetically coherent group, with the exception of few genera.However, as stated by Santana et al. (2004), those characters could be overall similarities, i.e., simplesiomorphies and/or homoplasies within Majoidea.The larval morphology of Notolopas brasiliensis is in agreement with the zoeal characters proposed by Santana et al. (2004) that characterize Pisidae.In the first zoeal stage, the setation of the coxal and basial endites of the maxillule (excluding Pisoides), and the endopod of the first maxilliped (excluding Doclea) could be mentioned (Table 2).The character that could distinguish the second zoeal stage is: the presence of the exopod seta on the maxillule (excluding Pisoides) (Table 3).The megalopa presents no consistent morphological character among genera (Table 4).
were generally followed.Specimens of larval stages and a spent female crabs have been deposited at the NEBECC Decapod Larval Collection, Núcleo de Estudos em Biologia, Ecologia e Cultivo de Crustáceos, Department of Zoology -IB, Universidade Estadual Paulista, Botucatu, State of São Paulo, Brazil, accession numbers NEBECCLC # 00066 and 0078.Slides used in the description have been deposited at the Museu de Zoologia da Universidade de São Paulo, São Paulo, State of São Paulo, Brazil under register number MZUSP 17086.

TABLE 1 :
Species of the Pisidae with known larval descriptions, indicating source and stages described.* not included in comparison.

TABLE 2 .
Comparison of larval characters of the first zoeal stage for Pisidae genera.

TABLE 3 .
Comparisons of larval characters of second zoeal stage for Pisidae genera; see table 2 for definition of abbreviations.

TABLE 4 .
Comparisons of larval characters of the megalopa stage for Pisidae genera; see table 2 for definition of abbreviations.