The larval development of Pilumnoides hassleri ( Decapoda : Brachyura : Pilumnoididae ) reared in the laboratory , with a review of pilumnoidid systematics using larval characters

The genus Pilumnoides is an interesting taxon because its systematic position, based on adult characters, remains unclear. These xanthoid crabs have been related to the Carpiliidae, Goneplacidae and eriphioidea. P. hassleri A. milne edwards, 1880 lives in Brazilian, uruguayan and Argentinian coasts as far south as the magellan strait (southwestern Atlantic). Larvae of P. hassleri from females collected in the harbour of mar del plata were reared in the laboratory from zoea i to megalopa and the first larval stage, and described. The species passed through 5 zoeal stages and a megalopa. Larval characters were compared with the previous description of larvae from the southeastern pacific species P. perlatus and with species of Carpilius, Goneplax and Eriphia in order to review the relationships between these taxa.

stating that the family was related to the carpiliids.To add further systematic confusion, Števc ˇic ´ (2005) assigned the pilumnoididae to the eriphioidea.karasawa and schweitzer (2006) found no support for any of the above proposals, considering that the pilumnoididae warranted their own superfamily.most recently in systema Brachyurorum, Ng et al. (2008) also recognised the familial status of pilumnoididae and assigned it to the pseudozioidea Alcock, 1898.
There are eight species of Pilumnoides that live in both the intertidal and deep waters, usually attached to algae (Guinot and macpherson, 1987).Five species occur in the pacific and Atlantic coasts of America, two in west Africa and one in england (Guinot and macpherson, 1987); the latter has been discovered "attached to hull of ships during the early part of this century but has not been found since" (ingle, 1997).Two species live in the southern end of south America, P. hassleri A. milne edwards, 1880 in the Atlantic, and P. perlatus (poeppig, 1836) rathbun, 1930 in the pacific.The latter is the type species of this genus.P. hassleri inhabits Brazilian, uruguayan and Argentinian waters as far south as the magellan strait, subtidally to 40 m depth; near mar del plata it has been found in dense aggregations (Boschi, 1964).P. perlatus inhabits coastal waters of perú and Chile, intertidally and subtidally to 54 m, and also reaches the magellan strait (retamal, 1981).
in march 2007 an ovigerous female crab of Pilumnoides hassleri A. milne edwards, 1880 was collected from mar del plata, Argentina, and the larvae were reared in the laboratory.The purpose of the present study is to describe the larval and first crab stages of P. hassleri and compare them with the morphology of P. perlatus, Goneplax, Eriphia and Carpilius in order to review the relationships among these taxa.mATeriAL ANd meThods ovigerous female crabs of Pilumnoides hassleri were collected by the authors in mar del plata harbour, Buenos Aires, Argentina on march 2007 and February 2008, transported alive to the laboratory, and maintained in an aquarium containing natural sea water until hatching.immediately after hatching, only actively swimming larvae were transferred with widebore pipettes to individual cultivation vials (25 ml) and cultured at 20°C, 33-35 psu, and an artificial light regime of 8:16 h (L:d).From zoea i to megalopa, Artemia sp.nauplii was offered as food ad libitum.Chaetoceros calcitrans and Brachionus plicatilis were added as food for zoeas i and ii.Water and food were changed daily, and larvae were checked for mortality and molts during each water change.
one female (march 2007) was used to obtain individuals of each stage for morphological studies, which were preserved in 4% formaldehyde.Another 4 females (February 2008) were used to estimate the intermolt period and the duration of the complete development on the basis of the culture of 20 newly zoeas i from each female, and to compare the size of zoea i among clutches.
specimens were dissected under an olympus sZ40 stereomicroscope.measurements and drawings were made using an olympus Ch30 compound microscope equipped with a camera lucida.The following measurements were made with a micrometer eyepiece (40X): in zoea larvae, carapace length (CL) from the base of the rostrum to the posterior margin and rostrodorsal length (rdL) from the tip of the rostral spine to the tip of the dorsal spine; in the megalopa stage, carapace length (CL) from the base of the rostrum to the posterior margin and carapace width (CW) as the maximum width.data of rdL and CL of zoeas i were tested for differences among clutches with a one-way ANovA.drawings and measurements were based on 5 larvae per stage.descriptions were arranged according to the standard proposed by pohle and Telford (1981) and Clark et al. (1998).six of the twelve long setae of the 1st and 2nd maxillipedes of zoea v (whole animal) were drawn.right pereiopods of megalopa (whole animal) were drawn, one to four, truncated.The aesthetascs of antennula and the long setae of all pleopods of megalopas were drawn truncated.
samples of larvae and the adult female were deposited in the museo Argentino de Ciencias  Naturales "Bernardino rivadavia" under the catalog numbers mACN-iN 37533. resuLTs The larval development included five zoeas and a megalopa.From 80 larvae cultured in February 2008, 16 (20%) reached the megalopa stage after 27.5 ± 2.22 days and one reached the first crab after passing 21 days as megalopa.Body measurement of zoeal and megalopal stages are summarised in Table 1.There was a statistically significant difference among clutches in the rostro-dorsal length, but not in carapace length, of zoeas i (ANovA, F = 9.50, P<0.001 and F = 1.117,P = 0.355, respectively).morphological features and setation formulae through zoeal development are shown in Table 2.
Telson (Fig. 2i).each fork with 2 lateral (one very small) and 1 dorsal spine.one additional pair of inner setae on posterior margin.otherwise unchanged.
Antenna (Fig. 5C).endopod bud reaching the length of exopod.exopod with 2 terminal simple setae of unequal length and one long subterminal finely denticulated and two unequal subterminal spinules.
First crab (Fig. 9) General view (Fig. 9).Carapace trapezoidal, CL/CW = 0.99.Frontal margin bilobed, each side with six teeth and strongly denticulated; six small teeth, four short, two medium and two long papose setae just behind the margin; supraorbital arc denticulated; postorbital tooth short; three small and three large lateral teeth; four groups of three papose setae each on latero-dorsal and on dorsal position; a group of latero-dorsal small protuberances; two fringes of very small protuberances at the border of posterior margin; surface setose as figured.Cheliped and first pereiopod with many protuberances and teeth.disCussioN

Comparison of the larval development of Pilumnoides hassleri and P. perlatus
The larval development of both Pilumnoides species described up to this moment (P.hassleri and P. perlatus) included 5 zoeal and one megalopal stages.several striking differences appeared when the zoeal morphology of these species was compared (Table 3).Both species had 4 carapacial spines but the lateral spines were smooth in P. hassleri and finely denticulated in P. perlatus; P. hassleri carried dorso-lateral knobs only on the 2 nd abdominal somite whereas P. perlatus had knobs on the 2 nd and 3 rd somites; the postero-lateral spines on the abdominal somites were smaller in P. hassleri; the number of pairs of setae on the posterior margin of the telson during the zoeal stages was 5 in P. hassleri and 6 in P. perlatus; and only 2 outer spines were observed in the telson of P. hassleri from zoea iii to v (instead of 3), although the first zoea of both species has 3 spines.
The morphology of megalopae of P. hassleri and P. perlatus is very similar in spite of the differences observed between the zoeal stages.The main observed differences were the endopod of the antennule (unsegmented in P. hassleri and 2-segmented in P. perlatus), the number of marginal setae of the scaphognathite and the number of natatory setae of the exopod of pleopods, which is higher in P. perlatus.
rice (1980) listed the characters that the "ancestral zoea of the higher Brachyura" must have had and suggested several evolutionary trends that consisted in a reduction or loss of features such as the carapace spines, abdominal structures and/or cephalothoracic appendages, from the antennule to the second maxilliped.Later in the same paper, taking into account these hypothetical trends, he compared the morphology of larvae at a familial level (from those families that were recognised at that time on the basis of adult morphology).interestingly, when the zoeal morphology of Pilumnoides hassleri and P. perlatus were compared considering the trends proposed by rice (1980), it resulted that the pleon of P. hassleri consistently had several derived characters: "a reduction in the number of abdominal somites carrying dorso-lateral knobs" (from 2 to 1), "a reduction of the size of postero-lateral spines on the abdominal somites", "a reduction in the number of posterior processes added to the telson during the zoeal stages" (from 6 to 5 pairs), and "a reduction or loss of the outer telson spines" (only 2 spines in zoea iii to v). meanwhile, the lateral spines are smooth in P. hassleri and finely denticulated in P. perlatus.evolutionary trends have not been postulated for the megalopa, a highly specialised stage adapted for the transition between planktonic zoeas and benthic adults and probably subjected to different selective pressures than those stages.Consequently, "the pattern that seems to be emerging from attempts to employ the megalopa as an aid to systematics and phylogeny is a disappointing one" (martin, 1988).some meristic differences observed between P. perlatus and P. hassleri could be related with size differences between species: both zoeal and megalopal carapaces are larger in P. perlatus and the number of setae of the scaphognathite of zoeas and megalopae, and of pleopods of megalopae, are higher in this species.When the complete larval development of P. hassleri and P. perlatus, both reared at 20ºC, was Table 3. -difference between the zoeal morphology (all stages) of Pilumnoides hassleri and P. perlatus (described by Fagetti and Campodonico, 1973).
There is probably a relationship between larval size and developmental time.Finally, both species showed morphological abnormalities at the end of zoeal development, probably due to artificial rearing conditions.

Contribution of larval morphology to the knowledge of Pilumnoides relationships
on the basis of adult morphology, the genus Pilumnoides was related to the Carpiliidae (Guinot and macpherson, 1987;karasawa and kato, 2003), the Goneplacidae (davie, 2002), the eriphoidea (Števc ˇic´, 2005), and the pseudozioidea (Ng et al., 2008).however, it was also proposed that this genus belongs to a monogeneric clade that is a sister-group of the large clade that included the Xanthoidea, eriphioidea, progeryonoidea, Goneplacoidea and portunoidea (karasawa and schweitzer, 2006) rice ( 1980) addressed the question of the role of zoeal morphology on Brachyura classification.Considering that "adult crabs exhibit adaptations for a wide range of life styles" and that "the zoeal stages of all crabs are adapted to the same mid-water habitat", he concluded that groupings based on larval characters "should be largely free" from failures to recognise convergence among adult forms (rice, 1980).several attempts to group zoeas of the former family Xanthidae (now xanthoid sensu lato) were made (see rice, 1980 and references therein), and finally martin (1984) proposed a classification based on 6 groups.Pilumnoides perlatus was included by this author in group iii, together with species from the current eriphioidea and Xanthoidea (sensu karasawa and schweitzer, 2006), characterised by "Antennal exopod robust, about 1/2 length of protopod, armed with 3 unequal terminal setae.some characters shared with Group i (setation of endopod of maxillule and maxilla 1-6 and 3 + 5, respectively; basal segment of endopod of second maxilliped with 1 seta).other characters variable.usually 4 zoeal stages" (martin, 1984).on the basis of the present results, P. hassleri zoeas should also be included in group iii.Later, martin (1988) reviewed the morphology of xanthoid megalopae and performed a numerical phenetic analysis.he observed that "the resulting phenogram does not strongly reflect previ-ous groupings of adults or zoeas".since 1984, the number of descriptions of the complete larval development of xanthoids has continuously increased and the systematics of these crabs, based on adult morphology, has been dramatically modified (e.g. karasawa andschweitzer, 2006, Ng et al., 2008).however, a new attempt to group larvae of xanthoids and to compare the results with adult classification is still lacking.
The morphology of the antennal exopod of Carpilius corallinus zoeas allows this species to be placed in group iii of martin (1984), since it is less than half as long as the protopod and bears 3 setae of unequal length on its apical end.however, the position of these exopodal setae is rather different in C. convexus and C. maculatus, which have one subterminal and two terminal setae, as occurs in P. hassleri; in addition, they are setose in C. convexus.The complete zoeal development of Carpilius has only been described for C. corallinus and includes five zoeal stages.it is characterised by "enormous size of the zoeas, especially the advanced stages, which seem to be the largest among xanthid zoeas, and perhaps one of the largest among brachyurans" (Laughlin et al., 1983).many morphological features of zoeas ii to v are strongly affected by size, making comparisons with Pilumnoides useless.The first zoea of C. corallinus differs from that of P. hassleri in having only 3 aesthetascs, in the presence of dorsoloateral spines directed ventrally on abdominal somites 3-4 and the absence of a dorsal spine on the telson furca (Table 4).
The morphology of first zoeas of C. convexus and C. maculatus is modified by a process of heterochrony.These species "appear to have hatched in a more advanced state of development than those of C. corallinus, and the expression of a number of characters has been accelerated (early onset)" and their development "appear to be abbreviated because the first zoeas are considered to be equivalent to the third-stage zoeas of C. corallinus" (Clark et al., 2005).disregarding those characters clearly affected by developmental abbreviation, the first zoeas of P. hassleri and C. convexus and C. maculatus differ in the relative length of carapace spines and the protopodal process of antenna and in the presence of dorsolateral knobs on abdominal somites 3-5.Finally, the telson of P. hassleri, C. convexus and C. maculatus has a dorsal spine on each fork.
The antennal exopod of Goneplax rhomboides is shorter than the rostral spine and acutely tipped; it has spinules about half-way along its length but the 3 terminal setae are lacking.in addition, the zoeal development of P. hassleri and Goneplax rhomboides differs in the number of antennular aesthetascs, in the number of setae of the distal segment of the endopod of the second maxilliped, in the presence of dorsolateral processes directed posteriorly on somites 3-4, and in the presence of only one lateral spine in the forks of the telson, even in zoea i (Table 5).otherwise, it differs in the number of stages (5 vs 4) and some characters related with this fact.
The complete larval development of E. gonagra, which includes 4 zoeas, has been described by Fransozo (1987) but a much more detailed description of the first zoea of E. scabricula has been published by Clark and paula (2003).The antenna of both species has 3 setae (1 long subterminal, 2 unequal terminal) in the exopod and appears to correspond to that of the group iii proposed by martin (1984).E. scabricula has two small lateral basal spines in the protopodal process of the antenna that were not observed in P. hassleri; however, both species share the presence of terminal spinules in the antennal exopod.otherwise, the first zoeas of P. hassleri and Eriphia scabricula differ in the relative length of carapace dorsal spine, in the setation of antennule (expopod), maxilla (coxal endite), and first maxilliped (coxa).Finally E. scabricula, but not P. hassleri, has teeth on the basial entite of the maxillule and dorsolateral processes on abdominal somites 3-5 (Table 6).
Based on morphological similarities and differences, P. hassleri zoeas are more similar to those of Carpilius, they differ in more aspects from those of Eriphia and differ in a key trait (antennal exopod) from those of Goneplax.These results reject the adult classification suggested by davie (2002) and support the opinion of Guinot and macpherson (1987) of a closer relationship with the Carpiliidae.however, Table 5. -difference between the zoeal morphology (all stages) of Pilumnoides hassleri and Goneplax rhomboides (Linnaeus, 1758) described by ingle and Clark (1983).

ACkNoWLedGemeNTs
The manuscript was substantially improved by reviews by p.F. Clark and G. Guerao.This paper was funded by grants from the universidad Nacional de mar del plata (Grant Nº 15e305), and the Agencia Nacional de promoción Científica y Tecnológica (ANpCyT, Grant Nº 21757) awarded to eds.NF acknowledges a doctoral fellowship from CoNiCeT.