Intraspecific variation in preserved specimens of the fiddler crabs Uca panacea and Uca pugilator ( Decapoda : Ocypodidae )

The fiddler crabs Uca panacea Novak & Salmon, 1974 and Uca pugilator (Bosc, 1802) are closely related North American species that are sympatric along the north coast of Gulf of Mexico. Since U. panacea was described, there has been confusion in the identification these two species. Morphological differences between these two fiddlers have been pointed out in recent years, mainly regarding the presence of a pigment spot and granulations on the dorsal margin of carapace in U. pugilator. We report herein some intraspecific differences between the two species that we believe to be useful in avoiding misidentification among preserved specimens of these two fiddler crabs, such as the absence of the pigment spot in U. pugilator and the presence of the pigment spot and granulations on dorsal margin of carapace in U. panacea as well. Our results have also revealed that 65% of the U. pugilator specimens examined possess a gape pile in the major chela, which is not a reliable diagnostic character, but could be useful when present. The gonopods of both species were analyzed using SEM, confirming the previous statement that the sub-terminal thumb is shorter in U. panacea than in U. pugilator. Finally, as important morphological characters are missing in the original description of U. panacea, a redescription of this species is also provided. KEY WORD. Distinctness; Gulf of Mexico; morphological characteristics; SEM analysis. 176 L. E. A. Bezerra & P. A. Coelho ZOOLOGIA 26 (1): 175–182, March, 2009 of the first ambulatory more numerously granulated in U. panacea than in U. pugilator. Moreover, the authors also noted a slight difference between the gonopods. However, the analysis of preserved species has revealed some intraspecific differences regarding those characteristics. Due to the difficulties in the reliable identification of these two species, the aim of this study is to provide information about intraspecific variability that we believe to be useful in avoiding misidentification among preserved specimens of these two fiddler crabs. A scanning electron microscopy (SEM) analysis of gonopods is presented in order to try to clarify the differences in gonopod morphology. Moreover, as some morphological features, especially in females, are missing from the original description of U. panacea, and standard taxonomic rules were ignored, a redescription of this species is provided. MATERIAL AND METHODS The fiddler crabs examined in this study are deposited in the National Museum of Natural History, Smithsonian Institution, Washington DC, USA (USNM) and in the American Museum of Natural History, New York City, USA (AMNH). Holotype and paratypes of U. panacea as well as additional material were examined and compared with U. pugilator specimens present in those collections. The gonopods of U. pugilator and U. panacea were examined using scanning electron microscopy. Gonopods used for SEM analysis were taken from a paratype of U. panacea (USNM 150089), and from specimens of both species collected inside and outside of the area of sympatry (U. panacea: USNM 171530; USNM 180188. U. pugilator: USNM 6440; USNM 55553; USNM 17186). The gonopods were prepared according to the methodology proposed by FELGENHAUER (1987). Abbreviations used in this study: (SEM) Scanning Electron Microscopy; (Co.) County; (m) male; (f ) female; (ovf) ovigerous females.


Intraspecific variation in preserved specimens of the fiddler crabs
Uca panacea and Uca pugilator (Decapoda: Ocypodidae) Luis E. A. Bezerra 1, 2 & Petrônio A. Coelho 1 1 Programa de Pós-Graduação em Oceanografia, Departamento de Oceanografia, Universidade Federal de Pernambuco.Avenida da Arquitetura, 50740-550 Recife, Pernambuco, Brasil.  2 Corresponding author.E-mail: luiseab@gmail.comABSTRACT.The fiddler crabs Uca panacea Novak & Salmon, 1974 and Uca pugilator (Bosc, 1802) are closely related North American species that are sympatric along the north coast of Gulf of Mexico.Since U. panacea was described, there has been confusion in the identification these two species.Morphological differences between these two fiddlers have been pointed out in recent years, mainly regarding the presence of a pigment spot and granulations on the dorsal margin of carapace in U. pugilator.We report herein some intraspecific differences between the two species that we believe to be useful in avoiding misidentification among preserved specimens of these two fiddler crabs, such as the absence of the pigment spot in U. pugilator and the presence of the pigment spot and granulations on dorsal margin of carapace in U. panacea as well.Our results have also revealed that 65% of the U. pugilator specimens examined possess a gape pile in the major chela, which is not a reliable diagnostic character, but could be useful when present.The gonopods of both species were analyzed using SEM, confirming the previous statement that the sub-terminal thumb is shorter in U. panacea than in U. pugilator.Finally, as important morphological characters are missing in the original description of U. panacea, a redescription of this species is also provided.KEY WORD.Distinctness; Gulf of Mexico; morphological characteristics; SEM analysis.
of the first ambulatory more numerously granulated in U. panacea than in U. pugilator.Moreover, the authors also noted a slight difference between the gonopods.However, the analysis of preserved species has revealed some intraspecific differences regarding those characteristics.Due to the difficulties in the reliable identification of these two species, the aim of this study is to provide information about intraspecific variability that we believe to be useful in avoiding misidentification among preserved specimens of these two fiddler crabs.A scanning electron microscopy (SEM) analysis of gonopods is presented in order to try to clarify the differences in gonopod morphology.Moreover, as some morphological features, especially in females, are missing from the original description of U. panacea, and standard taxonomic rules were ignored, a redescription of this species is provided.

MATERIAL AND METHODS
The fiddler crabs examined in this study are deposited in the National Museum of Natural History, Smithsonian Institution, Washington DC, USA (USNM) and in the American Museum of Natural History, New York City, USA (AMNH).Holotype and paratypes of U. panacea as well as additional material were examined and compared with U. pugilator specimens present in those collections.The gonopods of U. pugilator and U. panacea were examined using scanning electron microscopy.Gonopods used for SEM analysis were taken from a paratype of U. panacea (USNM 150089), and from specimens of both species collected inside and outside of the area of sympatry (U. panacea: USNM 171530; USNM 180188.U. pugilator: USNM 6440; USNM 55553; USNM 17186).The gonopods were prepared according to the methodology proposed by FELGENHAUER (1987).

TAXONOMY
Uca panacea Novak & Salmon, 1974 Figs 1-4, 8, 11-13 Gelasimus pugilator Stimpson, 1859: 62 in part;Smith, 1870: 136 in part.Uca pugilator Ortmann, 1897: 352 in part;Rathbun, 1900: 585 in part;1918: 400 in part;Fingerman, 1956Fingerman, : 274, 1957: 7;: 7;Rao & Fingerman, 1968: 27;Crane, 1975: 223 in part).Uca panacea Novak & Salmon, 1974: 313, figs 1-7;Powers, 1977: 53;Salmon et al., 1978: 252;Barnwell & Thurman, 1984: 41).Male redescription.Carapace: carapace moderately arched; front wide, contained about three times in width of carapace between antero-lateral angles.Antero-lateral margins slightly divergent, angling bluntly into dorso-lateral margin, which is slightly beaded.Postero-lateral stria short, faint, located immediately above 4 th ambulatories.H-form cardiac depression moderately outlined, colored rust-red.Dorsal margin of carapace without pile, but with fine granulations along antero-and dorso-lateral margins.Pair of small brown spots slightly anterior to H-form depression, two additional purple pigmentation spots near the base of front and two white spots externally to purple pigmentation, near upper margin of eyebrow.Orbits moderately oblique; eyebrow almost vertical, but well visible in dorsal view, breadth about half of diameter of adjacent part of depressed eyestalk, lower margin beaded.Suborbital margins with crenellations little developed internally, becoming more developed and separated along outer orbital margin, not obscured by setae or pile.Row of setae on floor of orbit, immediately above sub-orbital crenellations.All abdominal segments distinct, not fused.Pleonal clasping or lock apparatus present.Minor cheliped: merus slender, dorsal margin convex; antero-and postero-ventral margins straight.Carpus without tubercles or tuberculate ridge.Pollex and dactyl longer than palm; gap narrow, inner margins with few serration, long in proximal end, decreasing distally, not in contact; tip of dactyl and pollex with row of soft long hairs on ventral margins.Major cheliped: antero-dorsal margin of merus straight, arching near distal end; ventral margin straight, with blunt tubercles increasing in size distally.Antero-dorsal margin of carpus with row of tubercles, ending in strong, blunt proximal tubercle; inner margin with oblique tuberculate ridge formed by indistinct tubercles, almost absent near upper end.Outer manus covered by large tubercles, decreasing in size near ventral margin; longitudinal keel starting in distal third, at about middle manus, well above ventral margin, and extending along most of pollex on ventral half, poorly developed distally.Palm covered by larger tubercles, merging in small tubercles in lower region.Oblique tuberculate ridge absent, no tubercles along margin of carpal cavity.Upper margin of carpal cavity with pile in proximal end.Oblique pre-dactyl tuberculate ridge formed by larger tubercles, continuing downward along inner margin of pollex.Dactyl long and curved downward.Pollex straight, slightly turned upward, with serration along inner and outer margin.Row of tubercles in center of pollex, with enlarged teeth halfway to its tip, and other near the tip of pollex.Both pollex and dactyl slender and flatted.Gape pile absent.Intraspecific variation in preserved specimens of the fiddler crabs ZOOLOGIA 26 (1): 175-182, March, 2009 Ambulatories: merus slender, dorsal margin almost straight with row of setae on major side, and armed with short oblique rows of small tubercles on minor side; antero-and postero-ven-tral margins slightly convex, and beaded on minor side.Carpus and manus without serrations or rugosities.First ambulatory on major side with merus, carpus and manus roughened anteriorly Female redescription.Carapace: dorsal surface finely granulated, mainly near antero-and postero-lateral margins, which are both beaded.Postero-lateral stria long, beaded, located immediately above 4 th ambulatories.Suborbital crenellation stronger than in males, with two rows of setae on floor of orbit, immediately above sub-orbital crenellations.Abdominal segments not fused.
Minor cheliped: as in males.Ambulatories: merus slender as in males, without row of setae on dorsal margin, which are numerous and long in ventral margins.Setae in dorsal margin are sparse and short.Short oblique rows of tubercles in dorsal margin of merus stronger than in males, in both sides, as well as the serrations in antero-and postero-ventral margins.Carpus and manus rugose throughout; antero-dorsal margins of 3 rd and 4 th legs armed with serrations.
Gonopore: roughly triangular, not tuberculate.Remarks: this description was based on the holotype deposited at the USNM.The following remarks are based on the examination of paratypes and additional material.The color pattern in carapace of preserved specimens is very variable, being completely absent in some of them.The postero-lateral stria in some males is not as faint as in the holotype.One of the following characters can be absent in major cheliped in some specimens: the blunt tubercle in the antero-dorsal margin of carpus, the enlarged teeth in the inner margin of pollex, and the pile in the upper margin of carpal cavity.

Morphological variations
Regarding the differences between U. panacea and U. pugilator pointed out by BARNWELL & THURMAN (1984), some examined specimens of U. panacea present small tubercles on dorsal margin of carapace as in U. pugilator (Figs 1 and 2), as well as the branchial chamber, which is depressed relative to the central gastric region.Moreover, BARNWELL & THURMAN (1984) pointed out that a purple pigment spot may occur anterior to the H-form depression on the carapace of U. pugilator and not in U. panacea.However, this character is only good for live or recently preserved specimens, given that it does not persist in 70% ethanol.This pigment spot is easily confounded with another pigment present on the dorsal margin of carapace called gnathobases attachment, which may occur in both species.In 45% of the U. pugilator specimens examined, gnathobases attachment is absent (Figs 3-6).
The tubercles on the anterior surface of merus on the major side of first ambulatory in U. pugilator are less numerous compared to those in U. panacea (Figs 7 and 8).These tubercles are a diagnostic character between the species, confirming the previous statement by BARNWELL & THURMAN (1984).Some U. pugilator specimens possess a gape pile in the major cheliped (Fig. 9).The major chela was absent in 53 of the 734 males examined; so, in 681 males with major chela examined, the pile was present in 442 specimens (65%), and in  (35%) the pile was absent.Considering the areas of occurrence of both species (Fig. 10), along the US Atlantic coast, where U. panacea is absent, 358 out of 485 specimens examined had the pile (78%).Of the U. pugilator examined from south of Tampa Bay, 39 out of 120 (32.5%) had pile present.In the sympatric area (north of Tampa Bay) the pile was present in 45 out of 76 specimens examined (59.2%).
The females of both species have the dorsal region of carapace granulated.Females of U. pugilator bear large tubercles on the antero-lateral region, as previously observed by BARNWELL & THURMAN (1984).However, some of U. panacea females have tubercles on the dorsal region of carapace very similar to those of U. pugilator.Reliable distinction between single preserved females of these two species remains a difficult task.

SEM analysis of the gonopod
The SEM analysis has revealed that the distance from the base of the sub-terminal thumb to the tip of the gonopod in U. panacea is shorter than U. pugilator, as well as the thumb itself as observed by BARNWELL & THURMAN (1984).The length of the sub-terminal thumb in U. panacea is approximately one third of the length from the tip of the gonopod to the sub-terminal thumb base, whereas in U. pugilator the length is about half.However, when small specimens are analyzed, this difference is negligible (Figs 11 and 14).Thus, this is a very slight difference, being most conspicuous in large specimens (Figs 12 and 15), as also observed by BARNWELL & THURMAN (1984).We examined in detail the tip of the gonopod of both species and found no apparent differences (Figs 13 and 16).In contrast to the drawings provided by NOVAK & SALMON (1974), our analysis has revealed no differences on the curvature of the appendage.

DISCUSSION
Among the Gulf of Mexico fiddler crab species, U. pugilator and U. panacea are a typical case of cryptic species; although live specimens are clearly distinguishable by differences in color and behavior, only slight morphological differences separate these two species (MAYR 1963, Dr M. SALMON 2007, pers. comm.).According to M. SALMON (pers.comm.) the species occupy different niches and are sufficiently different in their courtship behavior to avoid any interbreeding.Hybrids of the species are not known.When the two species were forced to interbreed in laboratory by isolating males of one species with females of the other, most of the larvae died and those that survived were apparently infertile (SALMON et al. 1978).
However, according to VON HAGEN (1980), "nobody can fully estimate the degree of intra-specific variation of e.g.sound production in Uca, it is necessary to centre on morphological characters, the variations of which are far better known in many cases."The failure to account for intraspecific variation, allied to the inability of taxonomists to reach agreement about the status of some forms and the limitations of working with small number of specimens, sometimes poorly preserved (BARNWELL & THURMAN 1984), has been responsible for the description of non-valid species in recent years (e.g.U. virens Salmon & Atsaides, 1968, U. pavo George & Jones, 1982).
In addition to the morphological differences pointed out by BARNWELL & THURMAN (1984) between U. panacea and U. pugilator, the presence of gape pile was found in some U. pugilator specimens.However, it is important to keep in mind that presence/absence of gape pile is a variable character, and was only present in 65% of U. pugilator examined.CRANE (1975) speculated that the gape pile serves as a kind of buffer during the combat, masking noise or tactile sensations that might interfere with stimuli resulting from the rubbing of the gape tubercles along the predactyl ridges of the opponent.This kind of combat, named pregape-rub, was clearly observed three times, only in U. pugilator (CRANE 1975).The absence of gape pile in some specimens may be because the pile is fragile and easily dislodged, thus its occurrence is not specially mentioned in the systematic descriptions (CRANE 1975).Therefore, the pile could not be considered a reliable diagnostic character.On the other hand, this variable character may be useful in species identification.
The increase in the use of new morphological characters has been important to taxonomists in distinguishing very similar species, mainly regarding the Uca species from Gulf of Mexico, where many closely related species are found (BARNWELL & THURMAN 1984, ROSENBERG 2001, BEILINCH & VON HAGEN 2006).
The differences in gonopod morphology are useful only when large specimens are compared.Moreover, it is necessary to examine large series of specimens of several sizes to proceed to a confident distinction between these fiddlers based on gonopod morphology.VON HAGEN (1980) was unable to distinguish between the gonopods of the holotype of U. panacea and U. pugilator, probably because he examined specimens of U. pugilator of similar size of the holotype of U. panacea (approximately 16 mm of carapace width).At this size the differences in gonopod morphology are negligible.
In conclusion, the intraspecific variations found by us in preserved specimens of U. pugilator and U. panacea, as well as the additional morphological character in U. pugilator, allied to the SEM images of gonopods of both species, could help in identification of preserved specimens and avoidance of confusion between these two species.