﻿Evidence using morphology, molecules, and biogeography clarifies the taxonomic status of mole crabs of the genus Emerita Scopoli, 1777 (Anomura, Hippidae) and reveals a new species from the western Atlantic

﻿Abstract Uncertainties regarding the taxonomic status and biogeographical distribution of some species of the genus Emerita from the western Atlantic led to thorough examination of the subtle morphological differences between two coexistent species (E.brasiliensis Schmitt, 1935 and E.portoricensis Schmitt, 1935) along the Brazilian coast and compare them using two genetic markers. The molecular phylogenetic analysis based on sequences of the 16S rRNA and COI genes showed that individuals identified as E.portoricensis were clustered into two clades: one containing representatives from the Brazilian coast and another containing specimens distributed in Central America. Our molecular-based phylogeny, combined with a detailed morphological analysis, revealed the Brazilian population as a new species, which is described here as Emeritaalmeidai Mantelatto & Balbino, sp. nov. The number of species in the genus Emerita is now raised to 12, with five of them occurring in the western Atlantic, five in the Indo-Pacific, and two in the eastern Pacific.

These patterns of geographical distribution and uncertain records in the western Atlantic raise questions about whether these gaps are due to a lack of faunal surveys and/or a misidentification of specimens that are morphologically similar. Thus, we were motivated to perform a reassessment of the specimens assigned as E. portoricensis and E. brasiliensis along the Brazilian coast, using both morphological and molecular tools to evaluate the phylogenetic relationships between species of Emerita. We also examined the possible existence of cryptic taxa, which resulted in the new species described herein.

Sample collection and morphological data
Almost all specimens of Emerita analyzed herein were obtained by us and are deposited in the Crustacean Collection of the Department of Biology (CCDB) at the Faculty of Philosophy, Sciences and Letters at Ribeirão Preto (FFCLRP), University of São Paulo (USP), Brazil. Additional species of Emerita and other genera of Albuneidae (see Boyko 2002) were obtained and used in order to root the phylogenetic analyses. Individuals were collected by hand during low tide at different sandy beaches along the geographic distribution of the species (see references in Introduction). We also studied specimens obtained by means of loans or donations from University of Louisiana at Lafayette Zoological Collection, LA, United States (ULLZ -recently transferred to the National Museum of Natural History, Smithsonian Institution, Washington, D.C. USNM; old catalog numbers are used in the text).
Specimens were identified according to previous morphological characters established in the literature (Calado 1990;Melo 1999;Felder et al. 2023). All data along with new characters/variation were also considered for the comparative analysis along the species' geographic distribution. When secondary sexual characters (presence of the gonopores on the coxae of the fifth pair of pereopods and absence of mature pleopods for males -♂s, and the presence of the gonopores on the coxae of the third pair of pereopods and presence of mature pleopods or eggs for females -♀s) were not conspicuously observed, specimens were classified as juveniles (Delgado and Defeo 2006). Most of the morphological characters followed the references cited above and are designated in Fig. 1. Analyses were made and photographs were taken under a LEICA M205C stereomicroscope equipped with a LEICA DFC 295 camera, and measurements (mm) of structures were taken using the software Leica Application Suite.

Molecular data
The molecular markers 16S rRNA and cytochrome c oxidase subunit I (COI) were chosen because these mitochondrial genes are effective in studies that contribute to our comprehension of decapod diversity (see Schubart et al. 2000 andBracken-Grissom 2015 for references), including anomuran members (Mantelatto et al. 2006(Mantelatto et al. , 2009Miranda et al. 2020) and the target genus (Bhagawati et al. 2020(Bhagawati et al. , 2022. In this study, we used four different primers (see below). We used muscle tissue from the telson or 3 rd pereopods for DNA extraction according to the protocols proposed by Mantelatto et al. (2007) and Robles et al. (2007), and some adaptations were made to suit our material using the manufacturer's protocol of the salting-out method (Miller et al. 1988). The extracted DNA's final concentration was measured using a spectrophotometer (NanoDrop 2000(NanoDrop /2000c. Approximately 658 base pairs (bp) of the COI and 316 bp of the 16S rRNA genes were amplified using polymerase chain reactions (PCR) by thermal cycler (Veriti 96 Well Thermal Cycler Applied Biosystems). Fragments were amplified using the following thermal profiles: 16S rRNA -initial denaturing for 2 min at 94 °C; annealing for 40 cycles, 45 s at 94 °C, 45 sec at 46 °C and 1 min at 72 °C; final extension for 10 min at 72 °C; COI -initial denaturing for 2 min at 94 °C; annealing for 35 cycles, 30 sec at 94 °C, 30 sec at 50 °C, and 1 min at 72 °C; final extension for 7 min at 72 °C. We used the following primers: 16S-1472 (5'-AGA TAG AAA CCA ACC TGG -3') (Crandall and Fitzpatrick 1996) and 16SL (5'-CGC CTG TTT ATC AAA AAC AT -3') (Palumbi and Benzie 1991), HCO1-2198 (5'-TAA ACT TCA GGG TGA CCA AAA AAT CA -3') and LCO1-1490 (5'-GGT CAA CAA ATC ATA AAG ATA TTG -3') (Folmer et al. 1994). PCR products were observed in electrophoresis with 1.0% agarose gel and photographed with digital camera Olympus C-7070 on a UV transilluminators M20 UVP. Successful PCR products were purified using the SureClean Plus kit, following the manufacturer's protocol. Purified samples were sent to the Department of Technology at the College of Agricultural and Veterinary Sciences (FCAV, Jaboticabal) at São Paulo State University (UNESP) for sequencing.
A consensus was reached between the forward and reverse sequences of each specimen in BioEdit v. 7.0.5 (Hall 2005), and unspecific readings were manually corrected when required. Primer regions and non-readable parts at the beginning of the sequences were omitted. All consensus sequences were deposited in GenBank (http://www. ncbi.nlm.nih.gov/genbank/).
The alignment of the consensus of all sequences used in the phylogeny was performed with MAFFT (Katoh and Standley 2006) in the software Geneious 2022.1 (Kearse et al. 2012). Three maximum likelihood (ML) phylogenetic analyses were performed using the IQ-TREE program (Miller et al. 2010), one with the COI gene, one with the16S rRNA gene, and one using a concatenated alignment. The evolutionary model that best fit the data was determined by IQ-TREE according to the Bayesian Information Criterion (BIC) (Luo et al. 2010) and used for tree inference. The branch support was evaluated by ultra-fast bootstrap with 2000 pseudoreplicates.
16S rRNA reconstruction The automated alignment of 16S rRNA with 316 bp included 50 sequences of Emerita species. The phylogenetic tree, generated by ML analyses, indicated a clear separation of each species of Emerita (Fig. 2). Emerita brasiliensis consisted of a single clade, with all specimens assigned to this species, which was supported by bootstrap values of 96%. In this analysis, the closest relative of E. brasiliensis was E. rathbunae, although with low support (31%). All specimens of E. almeidai sp. nov. were clustered in a strongly supported clade (bootstrap values of 91%), which was the sister group of E. portoricensis s.s. from Central America (bootstrap values of 99%). Specimens of Emerita talpoida were split into two groups, one of them containing individuals from Florida (USA) and Mexico and the second one containing individuals from Massachusetts and South Carolina (USA). The positioning of a supposed "E. analoga" (AF425322) in this second group indicated a misidentification that should be fixed in the GenBank database. The phylogram positioned E. benedicti as a sister species of the clade composed by E. almeidai sp. nov., E. portoricensis, and E. talpoida, although with low support (60%). This major group, including E. almeidai sp. nov., E. portoricensis, E. talpoida, and E. benedicti, is the sister group of the clade composed of E. brasiliensis and E. rathbunae.
The clade containing E. holthuisi and E. emeritus, species from the Indo-Pacific, was positioned as a sister group of the major American clade mentioned above.
Emerita analoga, with reservations on the above-mentioned misidentified specimen, formed a single well-defined clade, with individuals from California (USA) and Chile, and was positioned as the sister species of all other species of Emerita used in the reconstruction, including members from the Americas as well as the Old World (E. emeritus and E. holthuisi).

Cytochrome Oxidase I (COI) reconstruction
The automated alignment of COI sequences with 658 bp included some sequences of Emerita species from GenBank. The phylogram also confirmed the clear separation of every species of Emerita (Fig. 3), including the strongly supported position of Emerita almeidai  sp. nov. Some differences were observed in the phylogenetic position of some of the species included in this alignment compared to that of the 16S rRNA alignment. For instance, E. rathbunae was recovered as the closest relative of E. talpoida instead of E. brasiliensis. Once again, despite the low number of specimens of E. talpoida, a clear division into two groups was recovered, with a few differences in relation to the 16S rRNA topology (the individual from Mexico was separated from the Florida one). The phylogenetic positioning of E. analoga was maintained as sister to all other species of Emerita included in this analysis.

Concatenated phylogram
The concatenated topology obtained for the 16S rRNA and COI genes (Fig. 4) recovered the main groups that were observed in the two separate analyses carried out for each gene. All specimens of Emerita almeidai sp. nov. were clustered together in a well-supported clade. The only specimen of E. portoricensis included in the analysis was well separated from other species. These two groups were recovered as sister species in a larger clade, as can be observed in the 16S rRNA and COI phylograms. Emerita rathbunae was recovered as the sister species of E. brasiliensis, and E. talpoida was recovered as the sister species of the clade composed by E. rathbunae and E. brasiliensis. The division of E. talpoida in two subclades was not observed because there were only two specimens of this species. However, subtle differences could be inferred by the long branches connecting the two specimens within this clade. Emerita benedicti was recovered as the sister species of all other species of Emerita in the analysis.

Taxonomy
Below we present the list of examined material and the description of the new species. A comparative image shows details about the general morphology of the seven species of Emerita from the Americas (Fig. 5) and a detailed comparison between E. brasiliensis and E. almeidai sp. nov. (Fig. 6) is furnished to complement the information. The updated distribution (Fig. 7) and a comparative analysis of the main characters of these two species and E. portoricensis was presented in Table 2. Emerita portoricensis -Efford, 1976: 178, 179;Calado 1990: 266, 268, 271;Tam et al. 1996: 490;Haye et al. 2002: 904 (non Emerita portoricensis Schmitt, 1935 (Calado 1990;Melo 1999;Felder et al. 2023;present study) (Efford 1976;Melo 1999;Veloso and Cardoso 1999;Felder et al. 2023) and the present study. Diagnosis. Carapace dorsally convex, 1.42-1.54× longer than wide, surface densely covered by microcrenulate rugae; most rugae elongate and continuous across carapace median line not forming rows or lines; 17 or more rugae crossing median line, rugae obsolete laterally on epimeral lobes. Front with three distinct subacute lobes consisting of rostrum and two lateral projections, rostrum visibly shorter than lateral projections. Antennular flagellum dorsal ramus with 30 articles. Antennal peduncle second article large, with three distal spines, median spine the longest, antennal flagellum with 74-104 articles. First maxilla proximal endite rounded, subcircular with margins visibly convex; endopodal palp wide, short, distal end upturned. Third maxilliped without exopod, endopod with merus distal inner margin projected into strong subtriangular lobe, lateral margins of merus sinuous, outer distal margin ending on acute angle. First pereopod merus large, inflated, broad truncate lobe on inferior margin of merus, carpus distal end with large spine, propodus ca. as long as dactylus; dactylus elongate, more than twice as long as wide, superior surface almost straight, inferior surface convex with low, moderate, and regularly spaced serrations, dactylus lined by long plumose setae and short spiniform setae or spinules, terminus of dactylus with single short spine, terminus subacute. Pleon with second pleonite larger than others, tergite as wide as carapace, sides of second pleonite forming wide flanges laterally, second and third pleonite with two pairs of rugae extending from junction with next pleonite almost to ventrolateral margin. Overall coloration olive grey, white laterally, rugae distinctly white in coloration, few thin white bars or stripes near posterolateral regions of carapace.

Superfamily
Description. Carapace (Figs 1A, 5D, 6B, 8A, 11, 12A) elongate, 1.42-1.54× longer than wide, subcylindrical, overall dorsally convex, highly convex transversely, slightly convex longitudinally; carapace surface densely covered by low transverse microcrenulate to microdenticulate rugae, many of which are continuously elongate, not forming proper lines or rows of rugae, many continuous across middorsal region on anterior and posterior portions of carapace, usually 17 or more rugae extending across postcervical middorsal line; pterygostomial region with ventrolateral rugae; rugae separating small, anteriorly curved ridges; anterior margin of broad epimeral lobe of carapace with serrated appearance due to presence of such ridges. Pterygostomial plates densely punctate, separated from carapace by post-gastric groove; low slightly rugose ridge extending from median portion almost to distal end of plate, parallel to carapace margin for most of its extension, slightly deflected inwards near distal end. Front (Figs 5D, 6F) with three subacute dentiform projections; median projection forming broad triangular or subtriangular rostrum surrounded by relatively long plumose setae, distal end of rostrum sharply pointed, rostrum visibly shorter than lateral projections; lateral projections subtriangular with concave sides proximally and straight sides distally, visibly longer than rostrum, also surrounded by relatively long plumose setae; rostrum and lateral projections separated by wide U-shaped sulcus. Anterolateral margins of carapace just to the side of frontal projections surrounded by short plumose setae. Transverse frontal groove parallel to front, mostly straight, slightly bent at lateral extremes. Cervical groove just anterior to midlength of carapace, crescent shaped with convex face facing posteriorly, slight anteriorly facing notch on cervical groove on carapace midline. Most rugae broken, obsolete or absent on lower broad epimeral lobe.
Eyes (Fig. 5D) swollen at end of very narrow and elongated peduncles, reaching anteriorly past distal portion of fifth antennal peduncle article when extended and past spines of second antennal article when retracted; ocular peduncles composed of three articles; first article arcuate, longer than wide, convex on internal face and concave on external face; second article deflected downwards, longer than first article; third article long, first third wider, other two thirds very narrow, widening near eye.
Antennules (Fig. 8C) short; antennular peduncle composed of three articles; first article wider than others, external surface with large dentiform projection near base of article; second article densely setose, trapezoid in shape, dorsal surface shorter, ventral surface longer; third article short, also trapezoid in shape, dorsal surface longer, ventral surface shorter; flagellum dorsal ramus longer, with 30 articles, ventral ramus shorter, with 12 articles.
Antenna (Figs 6J, 8D) long; antennal peduncle composed of five articles; first article trapezoidal, longer than wide; second article large, covered by sparse rugae, distal end with three large spiniform projections, median projection longest, dorsal and ventral projections ca. the same size as each other, sulcus extending across dorsolateral surface from proximal end to base of dorsal spiniform projection, microdenticulate ridge separating ventral projection from median projection, one row of setose rugae present on mesial ventral portion; third article inserted on lateral portion of second article, completely concealed by second article in lateral view, trapezoidal in shape, proximal portion rectangular in shape, distal portion triangular, short line of setae parallel to distal margin; fourth article dorsally convex, ventrally Y-shaped; fifth article elongate, slimmer near base, inflated distally, row of setae on ventral margin; flagellum long, composed of 74-104 articles with dense long setae ventrally in adult specimens, number of articles smaller in juveniles, first article longest, ~ 3× as long as other articles.
First maxilla (Fig. 9B) small; proximal endite loosely connected to rest of appendage, oval, flattened, lateral and distal margins surrounded by relatively long setae; distal endite elongate, narrow, distal end slightly wider, pin-shaped, margins lined by setae, setae on proximal internal side very long, median and distal setae shorter, setae on proximal and median external portions shorter, longer subdistally; endopodal palp nearly as wide as long, tip slightly hooked upwards.
First maxilliped (Fig. 9D) membranous; exopod larger, arched, composed of two articles; proximal article subrectangular, outer margin convex, inner margin concave, distal article subovoid, surrounded distally by many long plumose setae, outer margin proximally convex until ca. midpoint, where it becomes concave, inner margin convex throughout its extension; endopod minute, elongate, membranous, with small tuft of setae subterminally; distal endite crescent shaped, exceeding length of exopod first article, extensively covered by short setae on external surface, inner margin covered by dense long plumose setae, patch of relatively long plumose setae present on distal end.
Third maxilliped (Fig. 9F, G) lacking exopod; endopod with coxa wider than long, small subtriangular projection on proximal inner side, base-ischium minute, much wider than long, merus broad, sparse setose rugae present on outer face, inner face mostly smooth with one large and distinct ridge crossing merus from base-ischium junction to carpus junction, margins lined by short setae, long plumose setae distally, nearly twice as long as wide, proximal third of inner margin very rounded, convex, distal two thirds straight, large subtriangular projection on distal end of inner portion of merus overlying part of carpus and propodus, outer margin slightly concave proximally, convex distally, distal end of outer portion of merus straight, carpus short, subquadrate, distal margin and inner face covered by long setae, propodus long, slightly curved inwards, outer face smooth, inner face densely covered in setae, dactylus long, shorter than propodus, slightly curved inwards, distal end rounded, outer face smooth inner face densely covered in setae.
First pereopod (Fig. 10A) coxa subtrapezoidal, sparsely covered by setose rugae on external face, setae on ventral margin and distal end, ventral dentiform projection proximally; base-ischium longer than wide, minute, lined by setae ventrally, with two lobes separated by median notch, ventral margin surrounded by setae; merus large, subcircular, sparsely covered by setose rugae, superior margin convex, inferior margin convex proximally, extended laterally forming truncate lobe with straight or almost straight lat-  eral margin, small short sulcus on mesial portion of distal end of merus; carpus elongate, crossed by some oblique rows of setose rugae on distal ventral portion, three very distinct small perpendicular rows of setose rugae on dorsal surface separating article from large narrow distal spine, spine reaching to base of dactylus; propodus subtrapezoidal in shape, sparsely surrounded by setae, sparse setose rugae present, a transversal ridge running along most of ventrolateral portion of propodus, including distal process (Fig. 8B), dense short setae running along ridge, distal process long, subtriangular, strong oblique ridge running from dactylus junction to base of distal process, superolateral surface of distal process excavate, fits base of dactylus, long setae present on distal process; dactylus (Figs 5D, 6D, 8B) elongate, usually more than twice as long as wide, superior margin mostly straight, inferior surface convex, inferior surface moderately serrated from median portion to distal end, terminus of dactylus acute or subacute bearing one small spine, weakly arched oblique ridge across superior portion of dactylus, ridge originating near median portion of junction with propodus, running upwards towards superior margin, fusing with superior margin around median portion of dactylus, ridge lined by small setae, long plumose setae surrounding dactylus, small spiniform setae among them.
Second through fourth pereopods (Fig. 10B, C, D) similar in configuration. Second pereopod coxa subquadrate; base-ischium small, longer than wide, surrounded by setae; merus large, subrectangular, longer than wide, surface covered by sparse setose rugae, superior margin mostly straight, convex towards distal end, inferior margin slightly concave, large dentiform projection protruding ventrally from distal end of merus, inferior margin lined by setae; carpus subtriangular, external surface with two short ridges present, one on superior and one on inferior regions of article, internal surface setose, crossed mesially by single row of setae, inferior portion with small triangular projection distally lined by setae; propodus wider than long, subrectangular, superior margin oblique, external face with small triangular projection positioned distally on dorsal region overlying part of dactylus, transverse ridge near superior margin, internal face with large spiniform projection lined by large setae at ca. same position; dactylus large, flattened, hook-shaped, broad proximally, narrowing distally, superior margin concave, inferior margin convex, distal tip upturned, inferior margin surrounded by long setae. Third pereopod coxa and base-ischium similar to second pereopod; merus subrectangular, much longer than wide; carpus very large, subtriangular, superior and inferior ridges present, propodus wider than long, subrectangular, superior margin oblique, small ridge near superior margin present, small triangular projection positioned distally on superior margin overlying part of dactylus; dactylus large, hook-shaped, flattened, broad proximally, narrowing distally, superior margin concave, inferior margin convex, distal tip upturned, inferior margin surrounded by setae. Fourth pereopod coxa and base-ischium similar to that of second and third pereopods; merus elongate, much longer than wide, subrectangular; carpus large, longer than wide, inferior surface almost straight, superior surface convex; propodus subquadrate, nearly as wide as long, lacking triangular projection, line of short setae near superior margin; dactylus large, somewhat flattened, broad distally, narrowing towards distal end, proportionally smaller than in other pereopods, subtriangular, superior and inferior margins almost straight, tip not upturned, a line of short setae parallel to superior margin, inferior margin lined by setae. Fifth pereopod (Fig. 10E) reduced, concealed under carapace; all articles except for dactylus elongate, much longer than wide, with small tufts of setae distally; propodus long, with distal projection that along with dactylus forms a small chela; dactylus short, deflected inwards; chela small, covered by setae.
Pleon short, partly recurved under carapace. First pleonite smallest, minute, much wider than long, fitting into posterior concavity of carapace; second pleonite larger than others, as wide as carapace, median portion of pleonite narrow, both sides of pleonite enlarged, forming two wide lateral flanges, flanges with pair of long transverse rugae extending from third pleonite junction almost to ventrolateral margins of tergite, distal portion of ventrolateral region of each pleonite with short transverse ruga extending from superior margin to inferior margin of narrowest portion of flange, wide lateral flanges forming space where third pleonite fits; third pleonite smaller than second, sides of pleonite somewhat enlarged forming flanges that are mostly covered by flanges of second pleonite, two transverse rugae extending from junction with fourth pleonite to junction with second on each flange; fourth pleonite smaller than third, sides slightly enlarged forming flanges which are mostly covered by flanges of third pleonite, one oblique ruga extending from junction with fifth pleonite to junction with third on each side of pleonite; fifth pleonite smaller than fourth, lateral flanges small; sixth pleonite subpentagonal, lateral margins forming subtriangular projections, two short longitudinal grooves near articulation with telson, each groove joined to two much smaller transverse grooves. Female pleopods on second through fourth pleonites developed as three long and narrow articles, not developed on first and fifth pleonites; males without developed pleopods on first through fifth pleonites; uropods large, protopod subrectangular, endopod suboval, rounded, distal margin densely covered in setae, exopod suboval, more elongate, distal margin densely covered in setae.
Telson (Figs 6H, 8E) lanceolate, lateral margins setose, slightly convex proximally, very slight notches at ~ 3/4 of length of telson, two short longitudinal grooves near junction with pleon, two long longitudinal ridges parallel to lateral margins of telson, distal end of telson subacute.
Coloration in life. Carapace overall olive grey dorsally, lateral regions white, rugae extending across carapace white, posterolateral regions of carapace with few slim white longitudinal lines or small white blotches; lines and blotches usually restricted to posterolateral region, but some specimens possess one white longitudinal line along posterior 1/4 of carapace median line. Pleonal somites olive-grey anteriorly, white posteriorly, forming a pattern of alternating olive-grey and white stripes (Fig. 12A).
Habitat. Shallow infaunal, lives in wave swash zone of sandy beaches or shallow subtidal sandy flats where it burrows shallowly in sand, moves with tidal rise and fall.
Etymology. The species name honors Alexandre O. Almeida, a valued friend and respected colleague who has contributed extensively to increase knowledge of the decapod crustaceans of Brazil.
Remarks. Emerita almeidai sp. nov. is closest to E. portoricensis and thus shares a wide range of morphological similarities, which is why for many years several specimens from Brazil were wrongly assigned to E. portoricensis (see Introduction). Both species have a carapace densely covered by microcrenulate rugae (Figs 1A, 5D, G, 6B, 8A, 11, 12) distributed in similar patterns, a front with three subacute lobes with the rostrum being distinctly shorter than lateral projections (Figs 5D, G, 6B), first pereopod dactyli more than twice as long as wide and not as rounded as in other species such as Emerita brasiliensis and Emerita talpoida (Figs 5, 6C, D), two pairs of rugae extending onto lateral flanges of the first two pleonites. However, some characters such as the carapace length and width ratio (cw./cl.), the number of articles on the antennal flagellum, the first maxilla, the dactylus of the first pereopod, and the coloration in life can be used to distinguish between these two species. The carapace in E. almeidai sp. nov. (Figs 1A, 5D, 6B, 8A, 11, 12A) tends to be more oblong than that of E. portoricensis (Figs 5G, 12B), usually being 1.42-1.54× as wide as long in adult specimens (vs. 1.49-1.64× in E. portoricensis, present study), although there is some overlap. The number of articles on the antennal flagellum of E. almeidai sp. nov. (Fig. 8D) also tends to be more variable than that of E. portoricensis, varying from 74 to 104 articles in adult specimens, while E. portoricensis usually has 76-86 (Felder et al. 2023;present study). Although there is still some overlap, this character is still useful to distinguish between the two species. However, juvenile specimens (see details in Materials and methods) may have many fewer articles on the antennal flagellum in both species, and thus this character is only useful for adult specimens. The first maxilla of E. almeidai sp. nov. (Fig. 9B) also differs from that of E. portoricensis (Felder et al. 2023: 349, fig . 3): in E. almeidai sp. nov. the proximal endite is wider, rounder and with more convex margins, and the endopodal palp is proportionally wider and shorter. The first pereopod dactylus (Figs 5D, 6D, 8B) is also distinct, with E. almeidai sp. nov. having low, moderately and regularly spaced serrations on the inferior surface of the dactylus, while E. portoricensis has a slight and irregular serrations, which in many cases can be absent. The coloration of these two species can also be used to distinguish between live specimens. As shown in a recent redescription of E. portoricensis (Felder et al. 2023: 345, fig. 1) and in the present work (Fig. 12B), this species has very wide white bars on the posterolateral regions of carapace along with a wide white bar along the posterior 1/4 of the carapace median line. Although E. almeidai sp. nov. shares some of these characteristics (Fig. 12A), the white bars are usually slimmer and the white bar along the carapace median line is usually absent; however, it was observed only in one freshly collected paratype specimen (MZUSP 43536). The white colored rugae, which were observed in all of the freshly collected specimens of E. almeidai sp. nov. (Fig. 12A), however, are not present in either of the specimens shown in the recent redescription of E. portoricensis (Felder et al. 2023: 345, fig. 1) or in the specimen analyzed in this study (Fig. 12B), suggesting that this character might be unique to E. almeidai sp. nov. The southernmost record for E. portoricensis (Venezuela and Trinidad) and the northernmost record for E. almeidai sp. nov. (Maranhão, Brazil) are very far apart and there is a strong marine barrier, the Amazon-Orinoco plume (see Curtin 1986 for physical characteristics) that can promote some isolation between northern and southern decapod populations (see Peres et al. 2022), and this is possibly the reason there are no records of these species coexisting in the same environment.
Emerita almeidai sp. nov. has been observed to co-occur with E. brasiliensis in Praia de Iriri, in the state of Espírito Santo, Brazil (CCDB 3992 and 7226), with specimens of both species being collected at the same locality and on the same day. The distribution of these two species overlaps along the coast of the states of Espírito Santo and Rio de Janeiro, and it is possible that they co-occur in more locations in these states. Emerita almeidai sp. nov. can be distinguished from E. brasiliensis by the shape of the dactylus, which is elongated and has a serrated ventral margin in E. almeidai sp. nov. (Figs 5D, 6D, 8B) and ovate and non-serrated in E. brasiliensis (Figs 5C, 6C). The dactylus length and width ratio (dl./dw.) is also a robust parameter to distinguish the two species, especially in individuals of similar size. In E. almeidai sp. nov., the dactylus is proportionally longer and narrower than that of E. brasiliensis. The front is also different (Figs 5C, D, 6C, D): in E. brasiliensis the lateral projections and the rostrum are ca. as long as each other. The patterns of distribution of the microcrenulate rugae are also distinct between these two species (Figs 5C, D, 6A, B), with E. almeidai sp. nov. having very dense and non-broken rugae across much of the carapace, while E. brasiliensis has rugae that  are more broken into cusps (Felder et al. 2023). Furthermore, the cw./cl. ratio is also useful to distinguish these two species since E. almeidai sp. nov. has an overall longer and narrower carapace when compared to E. brasiliensis. The antennae (Figs 6I, J, 8D) is another character that can be used to distinguish between the two species, because E. almeidai sp. nov. has 74-104 articles, while E. brasiliensis has 103-134 articles; however, there is a small overlap. The differences between the telson measurements obtained between the telson length and width ratio (tl./tw.) showed a tendency for telson growth in E. almeidai sp. nov. in relation to the increase in carapace length, while in E. brasiliensis this ratio tends to remain stable with increasing carapace length. Melo (1999) did not highlight significant differences for this structure, but Calado (1990) described the telson of E. brasiliensis as being lanceolate, larger than the pleon, with all margins with short bristles, and that of E. almeidai sp. nov. with a triangular shape, larger than the pleon, wider in the proximal portion, with margins also supporting short bristles, which corroborates the biometric data found in our analyses.
The other species of Emerita found in the western Atlantic Ocean, E. talpoida and E. benedicti, are not known to co-occur with E. almeidai sp. nov.; Emerita talpoida can be distinguished from the new species by the rounded and ovate dactylus of the first pereopod (Fig. 5D, F), while E. benedicti (Fig. 5B) has a very acute terminus of the dactylus compared to a more subacute and slightly rounded terminus for E. almeidai sp.
nov. The morphology of the first pereopod dactylus can also be used to distinguish the new species from other congeners in the Indian Ocean, Indo-Pacific and eastern Pacific.
Previous descriptions of mouthparts of species of Emerita are scarce, only existing for two species, E. talpoida and E. portoricensis (see Snodgrass 1952 andFelder et al. 2023). Thus, comparative studies of the mouthpart morphology of Emerita are lacking and could be of great importance. At least for E. almeidai sp. nov. and E. portoricensis, it has been noted that the morphology of certain articles of the mouthparts, in this case the first maxilla, can be successfully used to distinguish between species. Thus, future descriptions and redescriptions of species of Emerita should include such characters, which might be valuable for comparative taxonomic studies.
The number of species in the genus Emerita is now raised to 12, with five occurring in the western Atlantic (E. almeidai sp. nov., E. benedicti, E. brasiliensis, E. portoricensis, E. talpoida), five in the Indian Ocean or Indo-Pacific (E. austroafricana, E. emeritus, E. holthuisi, E. karachiensis, E. taiwanensis) and two in the Eastern Pacific (E. analoga and E. rathbunae). The actual number might even be higher, given that there is a large distribution hiatus between the populations of E. analoga from North and South America and genetic differences between the northern and southern populations of E. talpoida. The record of E. brasiliensis from Venezuela is doubtful and may be a misidentification or may represent a separate species given the large geographic hiatus (Fig. 7). All of these cases require a thorough study, as made herein for some congeners, to determine whether these actually represent different, yet very similar, species.

Discussion
The combination of morphological and molecular methods confirms the validity of each species of Emerita included in the analysis and showed a clear division of E. talpoida into two subgroups that should be studied in the future. In addition, our phylogenetic trees based on two molecular markers confirmed the presence of a cryptic species previously misidentified as E. portoricensis, which we described in detail as E. almeidai sp. nov. For more than eight decades, a group of Brazilian specimens of Emerita was treated as E. portoricensis by several authors (see Introduction). The distribution of E. almeidai sp. nov. from Maranhão to Rio de Janeiro (Brazil) in combination with the redescription of E. portoricensis by Felder et al. (2023) answered questions that were raised in the past by some authors (Efford 1976;Calado 1990;Melo 1999) about the gap/discontinuity that existed in the distribution of E. portoricensis. These questions were clarified by the recognition of two different species, one in each hemisphere along the western Atlantic.
Species in the genus Emerita are known to have relatively long planktonic larval stages (i.e., E. talpoida lasting 30 days and E. rathbunae lasting 90 days, Efford 1970; E. holthuisi lasting 52 days, Siddiqi and Ghory 2006), thus favoring the chances of dispersion to suitable habitats. This extended larval development plays a critical role in governing the genetic structure, phylogeography, and dispersion of mole crabs of the genus Emerita (Dawson et al. 2011), factors which are also influenced and defined by other influences such as currents, transport effects, and sea level changes as observed in other groups (Doherty et al. 1995;Bohonak 1999;Cowen et al. 2000;Dawson 2001;Byers and Pringle 2006;Lester et al. 2007;Lessios 2008). Although the dispersal potential is high for species that present this larval profile (Palumbi and Benzie 1991), it does not always lead to strong gene flow, since physical or biological barriers can interfere with this dispersal process. Thus, disjunct populations can accumulate substantial genetic differences over time (Tam et al. 1996) that can result in the appearance of species that are not recognized as such due to the absence of studies covering all the different populations within the area of distribution as well as the lack of a representative set of specimens from these populations. The newly described species E. almeidai sp. nov. fits within this pattern, since it is a southern population that appears to be separated geographically by the Amazon-Orinoco plume, which has been shown to be an important physiological barrier for larval dispersal of many marine taxa, including decapod crustaceans (see Mandai et al. 2018 andPeres et al. 2022 for references and details).
There are some examples to support this hypothesis of separation for marine decapod crustaceans with a wide distribution along the western Atlantic, as noted in E. almeidai sp. nov. Using shrimps as an example, a recent study expanded the diversity of seabob shrimps of the genus Xiphopenaeus Smith, 1869 with descriptions of two new species (Carvalho-Batista et al. 2019), and a new species of Latreutes Stimpson, 1860 was described for a population from Brazil ). Considering the low number of integrative studies devoted to the huge diversity of decapods in the western Atlantic, these cases might not be exceptional, and we expect that a considerable number of cryptic and undescribed species may be revealed in the future.

Insights on the evolution of the genus
There are no known fossils that provide information about the evolutionary history of Emerita or the paleontological origins of the genus. Molecular clock-based studies have suggested that all species of the genus evolved before the mid-to late Pliocene, although no centers of origin or biogeographic scenarios have been suggested. The hypothesis that Emerita species evolved at least before the late Neogene was raised by Tam et al. (1996). Vicariance and dispersal events probably played an important role in the speciation of Emerita. Other ecological, physiological, and oceanographic processes likely contributed to the final geographic distribution of the populations that gave rise to the different species of the genus seen today. The expansion and colonization of new geographic areas with subsequent reduction of gene flow were probably the mechanisms by which most of these species originated (Haye et al. 2002).
Species of Emerita present a geographic distribution in disjunctive regions, apparently with separate conspecific populations and/or with species that may coexist (see Tam et al. 1996 andFelder et al. 2023 for reviews). In the Americas, Emerita analoga is one of two species that inhabit the Pacific coast, recorded in both hemispheres, while Emerita rathbunae is restricted to the tropical region. In the western Atlantic, Emerita talpoida is found from Massachusetts to Florida and also in the Gulf of Mexico; Emerita benedicti occurs mainly in the inner part of the Gulf of Mexico; Emerita portoricensis inhabits the tropical sandy islands and Central American mainland shorelines of the Caribbean Sea; Emerita brasiliensis is distributed along the coast of southern South America, and Emerita almeidai sp. nov. is endemic to the Brazilian coast. These examples demonstrate that the vast majority of these species have wide geographic ranges and, thus, disjunct populations can be naturally genetically isolated (Tam et al. 1996), especially when natural barriers are present.
Outside of the Americas, Emerita holthuisi has a very wide distribution, from the easternmost part of Africa to the southernmost part of India. It can also occur along the east coast of Africa, but so far there are few records for this region. Emerita emeritus overlaps with E. holthuisi along the western coast of southern India and also occurs in the Indo-Pacific on the eastern coast of India, Malaysia, Indonesia (Efford 1976), and possibly in between. In the present study, these two species grouped together in a separate clade from the one composed of specimens from the Americas. Emerita austroafricana can be found along the southern portion of the east coast of Africa, in Mozambique, Madagascar, and South Africa (Schmitt 1937;Efford 1976). The molecular phylogenetic analysis using the COI gene carried out by Haye et al. (2002) recovered E. austroafricana as the sister species of E. emeritus, although E. holthuisi, the sister species of E. emeritus recovered in their phylogenetic analysis with the 16S rRNA gene and in the present study, was not included in the analysis. This suggests a close relationship between these three species, but the precise topology of the clade formed by these taxa cannot be determined at present. Emerita karachiensis occurs on the west coast of the Indo-Pak subcontinent (Niazi and Haque 1974) and has not been included in any molecular analyses. However, Niazi and Haque (1974) mentioned morphological similarities between E. karachiensis and E. holthuisi, suggesting a close relationship between these species. Therefore, it is likely that this species is also part of the clade encompassing the Indo-Pacific species, although its exact position within this clade remains to be determined. Emerita taiwanensis is known only from two localities in Taiwan (Hsueh 2015) and has not been included in molecular phylogenetic analysis either. Hsueh (2015) suggested a close relationship between species that possess an acute and elongate pereopod 1 dactylus, such as E. portoricensis, E. benedicti, E. holthuisi, and E. karachiensis. However, as indicated by our phylogenetic analysis, this character does not seem to be phylogenetically informative, as it does not define any clades and appeared and was lost at least several times during the evolution of the species in the genus. Thus, the phylogenetic relationships of E. taiwanensis remain uncertain.
In the hypothesis proposed by Tam et al. (1996) on the evolution of the Emerita species from the Americas, it was suggested that E. analoga (Pacific species) was diverged from the other five New World species and was distant from E. rathbunae (the other Pacific species). Furthermore, E. rathbunae was clustered as sister species of the other species of Emerita found in the western Atlantic instead of with E. analoga that inhabits the Pacific coast. This hypothesis also suggests that Emerita species in the Americas evolved from an ancestral stock that was split into two branches, one leading to E. analoga and the other leading to the five remaining species. Our phylogenetic analysis corroborates this hypothesis, as E. analoga was recovered as the sister taxon to all other species of Emerita. Furthermore, it is likely that after splitting from E. analoga, the other populations of Emerita were divided into two groups, one that gave rise to the western Atlantic species and another that gave rise to the Indo-Pacific species. Consequently, with this scenario in mind, it can be assumed that the ornamentation of the second joint of the antennal peduncle present in E. analoga and some of the Indo-Pacific species (Schmitt 1937) is plesiomorphic, while the apomorphic condition is found in the clade composed of the other American species.
The biogeographic scenarios for the origin of E. analoga are not clear, and two hypotheses have been proposed (see references below): in the first, the genus Emerita originated on the western side of the Atlantic Ocean. If the center of origin is the Atlantic Ocean, it can be assumed that the species currently distributed in the eastern Pacific (E. analoga and E. rathbunae) evolved from Atlantic ancestors that dispersed into the Pacific and became isolated due to the closing of the Isthmus of Panama. The isthmus was closed to surface marine water circulation ~ 3 Mya (Late Neogene) but closed to deep-water circulation much earlier (Malfait and Dinkelman 1972;Keigwin 1982). In the second scenario, the center of origin of the genus Emerita was the Pacific Ocean. The Atlantic may have been colonized through the isthmus and taxa that differentiated in the Atlantic were likely to be ancestors of the species that later recolonized the Pacific, in this case E. rathbunae (Haye et al. 2002). At the end of the Cretaceous (~ 65 Mya), South America and Africa were completely separated (Dietz and Holden 1970). Although it is not possible to know how many species of Emerita inhabited the Tethys Sea before the complete separation of South America and Africa, it can be inferred that the separation of the continents was an important event in the speciation of these animals.
The clade composed of the northwest Atlantic species (E. benedicti, E. talpoida, E. portoricensis) and E. almeidai sp. nov. from the southwest Atlantic, as recovered in the 16S rRNA analysis, underwent extensive species diversification compared to the clade formed by E. rathbunae from the Pacific and E. brasiliensis, from the southwestern Atlantic, since the separation of the Pacific and Atlantic oceans ~ 3 Mya. This is consistent with other studies suggesting that the marine biota (mollusks, corals, and foraminiferans) of the western Atlantic were dramatically transformed ~ 2-3 Mya (Jackson and Budd 1996;Allmon 2001). Some hypotheses postulate that this change occurred as a result of the environmental disturbance associated with glaciation in the northern hemisphere and the formation of the Isthmus of Panama (Harrison 2004).
The closure of the Isthmus of Panama strongly affected ocean circulation, nutrient distribution, temperature, and salinity of the western Atlantic, and therefore had a significant influence on the evolution of marine fauna (Coates and Obando 1996;Allmon 2001). The flow of the Gulf Stream through the Yucatan Strait became more intense than it was before the closure of the Isthmus (Richards 1968) with an intense upwelling of cold deep waters (Stanley 1986). Therefore, this type of current may be a significant factor acting as a barrier to separate the group of species from the Gulf of Mexico from those located in the western side of the Atlantic, as observed in the E. talpoida clade, composed of specimens from Florida and Mexico in one group and individuals from Massachusetts and South Carolina in another group. There have been some suggestions of the occurrence of E. talpoida in Caribbean waters (see Felder et al. 2023, who say there is no confirmed evidence of this distribution). Previous usage of crabs as models of study (Felder and Staton 1994;Scheineider-Broussard et al. 1998) have identified the Florida Peninsula as a geographic barrier between the western Atlantic and Gulf of Mexico populations, raising awareness about the presence of cryptic species resulting from genetic isolation between populations distributed in these regions.