MORPHOLOGICAL REMARKS IN THE GHOST SHRIMP Callichirus seilacheri ( BOTT , 1955 ) ( DECAPODA , CALLIANASSIDAE )

Callichirus seilacheri (Bott, 1955) is considered one of the most common ghost shrimps in the intertidal zone of sandy beaches along the eastern tropical Pacific. The present study provides new observations on the morphology of C. seilacheri, based on the revision of abundant material collected along the Pacific coast of Central America, including specimens from the type locality (Playa Los Blancos, El Salvador) of this species. The new features of C. seilacheri include: carapace with low triangular rostrum, without setae on tip; pereiopod 1 highly dissimilar in adult males, but not in females and juveniles of both sexes; first pleopod sexually dimorphic; females with oval gonopores, each one of them on the ventral coxal segment of the third pereiopod, and extra genital pores on the ventral coxal segment of the fifth pereiopod, these latter non-functional (non connected with the ovaries). Apparently, the presence of extra gonopores in females of C. seilacheri is a vestigial character shared with other representatives of the same genus.


INTRODUCTION
The ghost shrimp species Callianassa seilacheri was erected by BOTT (1955) based on two female specimens collected at El Salvador, in the Pacific coast of Central America.Subsequently, MANNING and FELDER (1986) transferred this taxon to the genus Callichirus, in a revision of type material of three American representatives of this genus (Callichirus islagrande [SCHMITT, 1935], Callichirus major [SAY, 1818], and C. seilacheri).Interestingly, these authors pointed out that holotype of C. seilacheri correspond actually to one male specimen (p.441), against the first determination established by BOTT (1955) who identified it as a female (p.49).Afterwards, SAKAI (1999) corroborated that holotype of C. seilacheri is a female (p.62), thus confirming the determined in the original description of this species.Callichirus seilacheri is considered one of the most common ghost shrimps in the intertidal zone of sandy beaches along the eastern tropical Pacific (FELDER, 2001).This species is intensely harvested by local fishermen (up to 1,200 shrimps collected per day) to be used as bait for recreational fishing (HERNÁEZ and GRANDA-RODRÍGUEZ, 2015).The captures of C. seilacheri have increased during the last decade as result of depletion in other traditional resources of the Pacific coast of Costa Rica (e.g., penaeid and pandalid shrimps: WEHRTMANN and NIELSEN-MUÑOZ, 2009;coastal fishes: INCOPESCA, 2006).To the best of our knowledge, there is no management plan for this species in Costa Rica.
During more than half a century, C. seilacheri was considered the only representative of the genus Callichirus along the eastern Pacific (SAKAI, 1999).But HERNÁEZ et al. (2015), through a detailed morphological comparison between specimens from the Pacific coast of Central America and Chile demonstrated that actually the South American populations of C. seilacheri belong to Callichirus garthi (RETAMAL, 1975).Nowadays, it is considered that the distribution of C. seilacheri ranges from the Pacific coast of Mexico to Costa Rica (HERNÁEZ et al., 2015), in sympatry with one undescribed species of Callichirus, which has been often mentioned in literature (FELDER et al., 2003).
Part of the external morphology of C. seilacheri was illustrated by BOTT (1955) in his original work (p.48, figure 7a-g).Since then, other works on C. seilacheri have provided illustrations of the holotype (MANNING and FELDER, 1986: p. 441, figure 3a-f), or from specimens collected in the Peruvian coast (SAKAI, 1999: p. 63, figure 12c-f;SAKAI, 2005: p. 129;SAKAI, 2011: p. 422, figure 64f-h), the Pacific coast of Mexico (AYÓN-PARENTE et al., 2014: p. 7-9, figures 5a-g, 6a-d, 7a-j), and the type locality of this species (HERNÁEZ et al., 2015: p. 992, 994, 995, figures 2b, 3b,d,f,h,j, 4c,d).Most of these contributions, however, are questionable because of errors during the examination of type material and taxonomic identification of analyzed specimens.For instance, MANNING and FELDER (1986) confused the sex of the holotype, arguing that this specimen is a male with the first pair of pereiopods in a minor form instead of an adult female (p.441).On the other hand, several illustrations of C. seilacheri presented in SAKAIʼs contributions, actually, belong to C. garthi (HERNÁEZ et al., 2015), and AYÓN-PARENTE et al. (2014) stated to have doubts during the identification of the specimens of Callichirus, wherefore assigned the analyzed material to C. cf.seilacheri (p.379).
In this study, we are particularly interested in examining the external morphology of C. seilacheri, in order to describe new morphological characters that contribute to the taxonomical knowledge of this taxon.New features include refined observations on the carapace, pereiopods, male and female pleopods, and the genital apparatus in males and females.The results of this study will contribute to the knowledge of the morphology and sexual system in one of the most characteristic ghost shrimps of the American coast.

METHODS
Specimens of Callichirus seilacheri were extracted in July 2013 from their burrows using a hand-made yabby-pump (diameter, 77 mm; length, 100 cm) from the intertidal zone of Playa Los Blancos (13°19'38"N, 88°58'10"W), El Salvador, informed as the type locality of this species (Figure 1A-C).In addition, we collected between June and December 2012 specimens of C. seilacheri from the locality of Mata de Limón (09°55'12"N, 84°42'37"W), Pacific coast of Costa Rica (Figure 1D), and analyzed other specimens from the Costa Rican coast previously deposited in Museo de Zoología (MZUCR), Universidad de Costa Rica, Costa Rica (MZUCR 2156-01, 2246-02; for details see below).Size is expressed as carapace length (CL, from the postorbital margin to the posterior margin of the carapace), measured under a stereomicroscope with ocular micrometer and camera lucida or using vernier callipers (0.01 mm).The sex determination was based upon macroscopic features such as the clearly elongated carpus of the major chelipeds (males) and the presence of colored gonads in females; when these criteria did not allow a definitive sex determination, the location of the gonopores was revised, too (HERNÁEZ and WEHRTMANN, 2007).The genital apparatus in males and females was analyzed through observation of location and morphology of gonopores, including the dissection of some specimens to examine of internal morphology of reproductive system in females.Drawings were made with the aid of a camera lucida to highlight the main characters of each species.Terminology and abbreviations follow SAKAI (1999).Specimens collected during this study were deposited in the Museo de Zoología, Universidad de Costa Rica  and in the Scientific Collection of the Research Group in Crustacean Biology (CRUSTA), Universidade Estadual Paulista (CRUSTA 120001, 120002, 120003, 120004, 120005).

Remarks on genital apparatus of Callichirus seilacheri
Externally, the genital apparatus of C. seilacheri consists of prominent gonopores on the ventral coxal segment of the fifth pereiopod in males (Figure 4A) and oval gonopores on the ventral coxal segment of the third and fifth pereiopod in females (Figure 4B, 5A-C).Our observations indicated that additional gonopore is present both in juvenile and adult females of C. seilacheri.The further gonopore on the coxal segment of the fifth pereiopod in females is non functional or at least we did not notice any connection between the genital pore and the ovary.The dissection of several female specimens corroborates this last observation.Both male genital pore and female are covered by a thin membrane that impedes the enter of any particle of sediment to reproductive system.The female reproductive system involves paired ovaries, one ovary shorter than another, both visible through pleonal region (Figure 5A, D).The ovaries usually run from the posterior region of carapace to fifth abdominal somite (Figure 5A).A pair of oviducts link ovary to gonopores located on the coxal segment of the third pereiopod (Figure 5B, D), however, it was not observed a duct connecting the gonopore of the fifth pereiopod to ovary (Figure 5C).The gut consists of a simple tube that at the level of the abdominal region is arranged between the two lobes of the gonad, while the hepatopancreas or digestive gland involves paired lobes extending from posterior region of carapace to the second abdominal somite, below gut (Figure 5E).

DISCUSSION
The original diagnosis of Callichirus seilacheri by BOTT (1955) includes the presence of eyestalks with elongated distal projection, bilobed telson, first pereiopod with slim chela poorly dentate, palm of second pereiopod with rounded anterior margin, and third pereiopod leaf-shaped.These features are currently insufficient to separate C. seilacheri from the other four American species of the genus Callichirus because most of these are synapomorphies.For instance, C. garthi, C. islagrande and C. seilacheri share eyestalk with elongated distal projections and a bilobed telson (MANNING and FELDER, 1986: p. 440-441, figure 2a,f, 3a,f;HERNÁEZ et al., 2015: p. 992, figure 2a,b), while all above species, including C. major and Callichirus santarosaensis SAKAI and TURKAY, 2012, have the minor cheliped slender (MANNING and FELDER, 1986: p. 438, figure 1d; SAKAI and TURKAY, 2012: p. 747, figure 10e).Our study, for its part, improves the diagnosis of C. seilacheri proposed by HERNÁEZ et al. (2015), providing a series of new characters to distinguish to this species from other American representatives of Callichirus.Callichirus seilacheri is easily distinguished from the illustrations available in literature for C. major and C. santarosaensis by the presence of eyestalks with an elongated distal projection (MANNING and FELDER, 1986;SAKAI and TURKAY, 2012;respectively).This feature, even, may be used to separate C. seilacheri from the other undescribed Callichirusʼ species and reported for the Eastern Tropical Pacific (see introduction section).The similarities between C. seilacheri and C. islagrande are obvious from the illustrations available for both species (see MANNING and FELDER, 1986;HERNÁEZ et al., 2015).However, C. seilacheri differs from C. islagrande because the minor cheliped merus present denticulations on ventral margin and carpus is straight on flexor margin.Main morphological characters of each American species of Callichirus are shown in Table 1.
In taxonomy, the diagnosis is a brief description that covers the main features to distinguish one taxon from other taxa.The lack of a good diagnosis is not a minor detail in any taxon, because it makes difficult the identification of any specimen.Albeit trivial, this crucial aspect for the identification of one taxon is many times ignored, taxonomically inappropriate or simply is absent in the original description of a species.For instance, an appropriate diagnosis is available for C. garthi, C. santarosaensis and C. seilacheri (SAKAI and TURKAY, 2012;HERNÁEZ et al., 2015;present study).By contrast, this aspect is completely ignored in the description of C. islagrande and C. major (SCHMITT, 1935;SAY, 1818;respectively).Given the similarities among American species of Callichirus and the presence of undescribed species of this genus occurring in the American coast (e.g., at the Pacific coast of Nicaragua, FELDER et al., 2003), we believe totally necessary the publication of suitable diagnosis for C. islagrande and C. major.The former is morphologically similar to C. seilacheri, while the latter species, require an appropriate description because its large population along the Atlantic coast probably represent a species complex.
Another additional aspect observed in the present study is related to stability of certain morphological structures along the ontogeny of C. seilacheri.With the exception of the first pair of pereiopods, which changes morphologically after sexual maturity of males, the remaining structures of the body in males and females of C. seilacheri are well conserved along the ontogeny, in terms of shape and number of segments.For instance, the first and second pleopods are sexually dimorphic both in juveniles and adult.This characteristic also has been observed in specimens of C. garthi and C. major from the Chilean and Brazilian coast, respectively (HERNÁEZ, 2014).The identification of conserved features may be more relevant than those characters that change during the adult phase because the former allow identifying one taxon in juvenile and adult phase.Such argument is especially relevant when most of diagnoses are based exclusively in adult characters, as in Callichirusʼ species.
In this sense, a brief comment arises from the critical revision realized by FELDER and DWORSCHAK (2015) about the description of C. santarosaensis as a new species.These authors dispute the validity of C. santarosaensis, mainly because the new species is erected from a damaged specimen probably in juvenile phase.According to FELDER and DWORSCHAK (2015), most diagnostic characters of C. santarosaensis fit to the morphology of juvenile specimens of C. major (p.269, figure 2c-f).We are in agreement with this argument, but there are other characters figured by SAKAI and TURKAY (2012) that clearly differs between these two species.For instance, in C. santarosaensis the male second pleopod is biramous with the endopod underdeveloped (SAKAI and TURKAY, 2012: p. 747, figure 10h), while in C. major is biramous with the endopod and exopod notoriously developed (RODRIGUES, 1971: p. 196, figure 17).This character is morphologically conserved between juvenile and adult phase in members of Callichirus, therefore we believe that such feature may be used to separate C. santarosaensis from C. major.
Male and female of C. seilacheri can be distinguished from each other by the morphology of the first pair of pleopod (bi-segmented in males but tri-segmented in females), presence/absence of extremely dissimilar first chelipeds (highly dissimilar in adult males but not in females and juveniles from both sexes), and presence/absence of orange or dark red ovaries -depending upon developmental stage -(exclusively present in adult females).The last two features only can be used to separate males and females in adult stage.Presence of one further gonopore on the fifth pereiopod in females of C. seilacheri does not make difficult the sexual determination in this species when the criterion used is the morphology of the first pleopod (see Figure 3C, D).
Females in C. seilacheri showed two pair of gonopores: one pair functional on the coxal segments of the third pereiopod and an additional pair non functional on the coxal segments of the fifth pereiopod.The presence of gonopores on the fifth pereiopod in females of C. seilacheri, as has been described universally for males of most decapods (FELGENHAUER, 1992), suggests the existence of a testicular part of ovaries in the feminine gonad.Our observations, however, showed the contrary.There no testicular tissue in the feminine gonad and neither masculinization in females of this species.
Usually, specimens of decapods with gonopores on the third and fifth pereiopods have been called intersex, albeit the intersexuality and hermaphroditism are two terms usually confused in literature (FORD, 2012).The first is an abnormal condition, while the second a sexual system (CORREA and THIEL, 2003).The intersexuality is a condition in that one individual develops simultaneously male and female characteristics either externally (e.g., male and female gonopores) or internally within the reproductive organs (e.g., ovitestes).Presence of intersexual individuals is an abnormal condition within a population produced, among other factors, by endocrine disrupting chemicals (COLBORN et al., 1996), parasitism (RODGERS-GRAY et al., 2004) and genetic abnormalities (PARNES et al., 2003).Intersexuality condition non-guarantees the individual performance as male or female, or even both simultaneously.Inversely, the term hermaphrodite must only be used when transitional of intersexual forms form part of the 'normal' life history of the organism and not because of

CONCLUSION
In conclusion, diagnostic features of C. seilacheri mainly include: (i) antennular peduncle article 2 reaching as far as antennal peduncle article 4; (ii) male larger cheliped with ischium strongly curved on dorsal margin, with hook on ventral margin; (iii) fixed finger with cutting edge smooth; (iv) male Plp1 uniramous and composed of two articles.Callichirus seilacheri is a dioecious species with a marked sexual dimorphism along their ontogeny, which is mainly expressed through the morphology of first pair of pleopod.Despite females of C. seilacheri have extra genital pores on the ventral coxal segment of the fifth pereiopod, these latter non-functional.

Figure 1 .
Figure 1.Lateral and dorsal view of male (A) and female (B) individuals of the ghost shrimp Callichirus seilacheri, scale bar = 1 cm; intertidal at Los Blancos, El Salvador, type locality of C. seilacheri (C) and Mata de Limon, central Pacific coast of Costa Rica (D); the inset shows a burrow opening of the ghost shrimp C. seilacheri, scale bar = 5 mm.

Figure 4 .
Figure 4. Schematic representation of the genital apparatus of male (A) and female (B) individuals in the ghost shrimp Callichirus seilacheri, scale bar = 5 mm.P3-5, indicate pereiopod 3-5, respectively.The insets show real photographs on gonopores in male and female specimen of C. seilacheri.

Figure 5 .
Figure 5. Adult female with developed ovaries in the ghost shrimp Callichirus seilacheri, in dorsal view (A); female gonopores on the coxal segment of the third pereiopod, in ventral view (B); extra genital pore of female on the coxal segment of the fifth pereiopod, mesial view (C); oviducts connecting the gonad with the genital pores, in dorsal view (D); internal morphology of female, in lateral view (E).