A new anchialine Stephos Scott from the Yucatan Peninsula with notes on the biogeography and diversity of the genus (Copepoda, Calanoida, Stephidae)

Abstract Surveys of the anchialine crustacean fauna of the Yucatan Peninsula (YP), Mexico, have revealed the occurrence of calanoid copepods. The genus Stephos Scott, 1892, belonging to the family Stephidae is among the most frequent and widely distributed groups in anchialine caves but has not been hitherto recorded from the YP. Recent collections from an anchialine cave in an island off the northern coast of the YP yielded many specimens of a new species of Stephos. The new taxon, S. fernandoi sp. n., is described here based on male and female specimens. The new species is clearly distinguished from its congeners by the following characters: male left fifth leg with three terminal lamellae plus subdistal process, right leg with distal row of peg-like elements; female fifth leg with single long, acute apical process; genital double-somite with two rows each of 4 long spinules adjacent to operculum; legs 2-4 with articulated setae. The diversity of the genus shows regional differences; the Australia-Western Pacific region is the most diverse (eleven species), followed by the Mediterranean (seven species) and the Northeastern Atlantic (six species); only four species are known from the Northwestern Tropical Atlantic (NWTA). The morphology of the female fifth leg was examined to explore possible biogeographic trends in the genus; patterns suggest multiple colonization events in the highly diverse regions and a relatively recent radiation in the NWTA, characterized by anchialine forms. The introduction of stephid copepods in the region may be a relatively recent event derived from colonization of benthopelagic ancestral forms and subsequent invasion onto cave habitats. The new species appears to be linked to the strictly anchialine Miostephos.


Introduction
The primitive calanoid copepod families Epacteriscidae and Ridgewayiidae are the most representative and diverse copepods in anchialine and subterranean environments worldwide (Fosshagen et al. 2001). Other calanoid families with cave-dwelling species are the Arietellidae, Pseudocyclopiidae, Pseudocyclopidae, Fosshageniidae, and Stephidae (Boxshall et al. 1990;Suárez-Morales and Iliffe 1996;Jaume et al. 1999;Fosshagen and Iliffe 2004). The latter family contains marine hyperbenthic forms living in coastal waters and in anchialine habitats. It contains 4 valid genera of which Stephos T. Scott, 1892 is the most diverse, currently incorporating 31 species (Bradford-Grieve 1999;Boxshall and Halsey 2004;Moon et al. 2015;Boxshall and Walter 2016). Members of this genus have been reported from tropical to polar latitudes worldwide; Stephos has been recognized to frequently inhabit submarine and anchialine caves (Jaume et al. 2008;Kršinić 2012Kršinić , 2015. The remaining three stephid genera are relatively small, with a restricted distribution; together they contain a total of 6 species (Razouls et al. 2015(Razouls et al. , 2016. Previous reports of the family Stephidae from the Northwestern Tropical Atlantic (NWTA) include only a few species: Stephos deichmannae Fleminger, 1957 from surface plankton in the Gulf of Mexico (Fleminger 1957;Suárez-Morales et al. 2009), S. lucayensis Fosshagen, 1970, andS. exumensis Fosshagen, 1970, both from bottom samples of the Bahamas (Fosshagen 1970). The two known species of the genus Miostephos Bowman, 1976, M. cubrobex Bowman, 1976 from Cuba and M. leamingtonensis Yeatman, 1980 from Bermuda are other anchialine stephids from the NWTA (Razouls et al. 2015(Razouls et al. , 2016. The anchialine crustacean fauna of the Yucatan Peninsula (YP) of Mexico is widely recognized as highly interesting, with many endemic species (Yager 1987;Iliffe 1992;Mercado-Salas et al. 2013;Boxshall et al. 2014). Members of the Ridgewayiidae (Suárez-Morales and Iliffe 2005) and Epacteriscidae (Suárez-Morales et al. 2006) have been hitherto recorded in the YP, but there are no data on the occurrence of other anchialine calanoid families. During a biological survey of the crustacean fauna of an anchialine cave in the island of Cozumel, off the northeastern coast of the YP, many male and female specimens of copepods were collected. A first analysis revealed the presence of a calanoid tentatively identified as belonging to the family Stephidae. A detailed examination revealed that these specimens represent an undescribed species of the genus Stephos which is herein described in full and compared with its known congeners. The distribution and diversity of the genus in the NWTA is also analyzed.

Materials and methods
Specimens were collected on 6 July 2014 during a biological survey of an anchialine cave, Cenote Tres Potrillos, on Cozumel Island at 20°27'3.2"N, 86°59'14.4"W, Quintana Roo, Mexico. From a small pool at the cave entrance, a vertical shaft opens into a very large chamber with a halocline at 11 m. Beneath the halocline, sulfidic, fully marine water reaches a maximum depth of 38 m. A 40 m long passage at 12 m depth extends off the side of the main chamber (Mejía et al. 2008, fig. 3). Other anchialine crustaceans from this cave includes the shrimp Barbouria yanezi Mejía, Zarza & López, 2008, Agostocaris sp., and Procaris sp., the isopod Bahalana sp., and the amphipod Mayaweckelia sp. Plankton specimens were collected in the halocline with the aid of a conical plankton net (50 µm mesh size). The collected material was fixed and preserved in 100% ethanol. The copepods were sorted from the original sample and then transferred to glycerol. Specimens were prepared for taxonomic analysis by dissecting all appendages and light staining them with Methylene blue; the appendages were examined as temporary mounts in glycerine and sealed with Entellan® as permanent mounts. Drawings were prepared using a camera lucida mounted on an E-200 Nikon compound microscope with Nomarski DIC at magnifications of 1000X. Male and female specimens were prepared for SEM examination with a JEOL SM-6010 microscope at facilities of ECOSUR in Chetumal, Mexico. The process in cluded dehydration of specimens in progressively higher ethanol solutions (60, 70, 80, 96, 100%) and drying with a treatment with hexamethyldisilazane (HMDS). Terminology of the body parts and appendages followed Huys and Boxshall (1991). Body length was measured from the anterior margin of the cephalosome to the posterior margin of the caudal rami. The type specimens are deposited in the collection of zooplankton held at El Colegio de la Frontera Sur (ECOSUR), Chetumal, Quintana Roo, Mexico (ECO-CHZ) and in the National Museum of Natural History, Smithsonian Institution (USNM) (MSC, Maryland), United States. Original samples are deposited at the Universidad de Quintana Roo (UQROO) Campus Cozumel, Mexico.
Antenna ( Fig. 1E) biramous, with exopod longer than endopod. Coxa armed with one seta. Basis with two distal subequal setae on medial margin. Endopod 2-segmented, first segment long, cylindrical, with short seta inserted at 2/3 of medial margin; distal portion of terminal segment with two lobes, proximal lobe with 8 setae; distal lobe with single short, lateral seta plus five long terminal setae. Exopod indistinctly 7-segmented, first segment with one long seta, second segment longest, armed with three setae, one proximal, one medial and one on distal position. Segments 3-6 with 1, 2, 1, 1 setae, respectively. Distal segment with crown of three long, terminal setae, subequal in length and diameter.
Left and right antennules 24-segmented, lacking geniculation, slightly longer than in female when extended posteriorly; antennulary armature as in female. Mouthparts and swimming legs 1-4 as in female.
Fifth legs (Figs 3B, 4I-L) uniramous, asymmetrical. Left leg five-segmented, about as long as right counterpart; proximal segment widest of ramus, with inner margin expanded. Second, third, and fourth segments elongate, fourth with triangular plate on distomedial angle; distal segment with three terminal lamellae tapering distally plus subdistal subtriangular process, and with short seta inserted proximally on medial mar-  gin (Fig. 4J). Right fifth leg (Fig. 3B) four-segmented, first and second segments cylindrical, robust, unarmed. Third segment elongate and tapering. Fourth segment very slender and bifurcating distally into "C"-shaped structure furnished with 6-8 peg-like elements along bifurcation (Fig. 4L). End of subdistal process acute, opposite end with apical leaf-like expansion (Fig. 4K). Etymology. The new species is named after the Mexican carcinologist Dr. Fernando Alvarez (Instituto de Biología, UNAM, Mexico), who has significantly contrib-uted to the knowledge of the Mexican crustacean fauna, particularly from caves and anchialine habitats.
Based on the morphology of the male fifth legs, Bradford-Grieve (1999) divided the species of Stephos into four distinct groups. The new species S. fernandoi can be assigned to "group IV" by its possession of a male right leg 5 with a narrow fourth segment. Currently, this group includes nine species: S. pentacan thos Chen & Zhang, 1965 Moon et al. 2015).
The new species is the only one in this group with a right leg 5 ramus combining a distal segment (segment 4) with diverging processes set at right angles with acute tips plus a series of peg-like elements along the longest process (Fig. 4L). It differs from S. angulatus because in this species, the processes are both apically rounded and the segment lacks the peg-like elements observed in the new species. The left ramus has a similar structure in both species, with segment 4 bearing a distal lobular process (Bradford-Grieve 1999, fig. 8; Fig. 4L) and three distal lamellae, but the new species has an additional subdistal process (Figs 3B, 4J). In S. marsalensis, the distal segment of right male P5 is unbranched   fig. 4d), thus diverging from the bifid condition found in S. fernandoi; also, the left leg has five lamellate hyaline processes on the distal segment vs. only three such processes in the new species. The anchialine S. vivesi has a left leg with eight narrow lamellae and a relatively simple, spatulate distal segment of the right leg with two proximal processes (Jaume et al. 2008, fig. 2b-d), thus diverging from the pattern observed in the new species. The fifth leg of the new species differs from that of S. geojinensis in the number of lamellae on the distal segment of the left leg, three (Fig. 4J) vs. seven long plus 13 short lamellae, and right leg with distal segment bifurcate, with both branches subequally long (Fig. 3B) vs. outer branch extremely long, inner branch reduced (Moon et al. 2015, fig. 4d). The same kind of distally asymmetrical right fifth leg is present in S. tsuyazakiensis (Tanaka 1966, fig. 1o), thus differing from the new species. In S. morii, the right leg distal segment has a strong inner bulb-like process (Greenwood 1977, fig. 4g) which is absent in the new species; also, the left leg terminal segment has a long, distinctive spiniform process on subdistal position which is not present in S. fernandoi (Fig. 3B). In S. rustadi, the right leg distal segment is chela-like, with an expanded inner margin and the left leg is clearly shorter that its right counterpart and has three distinctive hook-like processes (Strömgren 1969, fig. 3f), thus diverging from the fifth leg structure of the new species. In S. penthacanthos, the right fifth leg has a spiniform process on the outer margin and the terminal segment is modified into a long, slender claw-like process, curved inwardly (Chen and Zhang 1965, fig. 20.5), thus differing from the spatulate distal segment described in the new species. In S. pacificus, the right fifth leg distal segment is relatively simple, represented by an elongate, narrow unbranched structure (Ohtsuka and Hiromi 1987, fig. 3f), different from the pattern observed in the new species, clearly branched (Fig. 3B).
The female fifth leg of S. fernandoi differs from that known in most species of Stephos, which has a medial seta and/or a row of spinules on the distal segment. It most closely resembles the fifth legs of the two species of Miostephos, M. cubrobex from Cuba (Bowman 1976, fig. 13) and M. leamingtonensis from Bermuda (Yeatman 1980, fig. 5), both with an attenuated unarmed distal segment. The new species differs in the genus characters (i.e. three-segmented female urosome, six-segmented left male fifth leg, reduced male right fifth leg strongly resembling the female fifth leg) (Bowman 1976). In species of Stephos, the female genital double-somite has widely different patterns of ornamentation on the ventral and/or lateral surfaces, including rows of spinules with both symmetrical and asymmetrical arrangements (Ohtsuka and Hiromi 1987;Bradford-Grieve 1999;Costanzo et al. 2000), lack of surface ornamentation, as in S. canariensis (Boxshall et al., 1990) or S. grieveae (Kršinić, 2015) or a highly modified, strongly asymmetrical somite as in S. exumensis (Fosshagen, 1970). The new species has a unique pattern combining a symmetrical genital double-somite with an ornamentation pattern represented by a set of 4 spiniform elements at each side of the genital operculum; this pattern has not been observed in any other species of Stephos.

Discussion
The distribution of primitive anchialine copepods (i.e., epacteriscids, misophrioids) in the Caribbean, Yucatan, the Canary Islands, and the tropical Pacific has been taken to indicate their Tethyan origin (Fosshagen et al. 2001;Boxshall and Jaume 2000;Boxshall et al. 2014). There are, however, groups whose invasion of cave habitats and subsequent isolation into anchialine waters is related to more recent events. During the Pleistocene, when the eastern coast of the YP emerged (Iturralde-Vinent and MacPhee 1999), some stygobionts which already were established in caves became isolated by the overlying freshwater lens. Cozumel Island is the most recently emerged land of the YP (Vázquez-Domínguez and Arita 2010) so the new species may represent a second-ary invasion and probably colonized recently the youngest epicontinental anchialine systems of the YP.
Stephids are in general benthopelagic or anchialine forms, strongly associated with the bottom communities, but some species are known from the plankton (Fleminger 1957;Kos 1972;Ohtsuka and Hiromi 1987;Costanzo et al. 2000;Zagami et al. 2000;Moon et al. 2015). Except for the planktonic S. deichmannae, all the stephids found in the NWTA are cave-dwelling anchialine forms (Fosshagen 1970;Bowman 1976;Yeatman 1980). The availability of cave habitats in the NWTA region has favored a highly endemic stephid fauna, likely younger and less speciose but comparable with that of the Mediterranean.
Pattern D was deemed as the most primitive group, followed by groups A and B, also with a lateral seta and an apically elongate or short segment, respectively. The derived pat- Figure 5. Geographic distribution of Stephos female fifth leg groups A-F. Group A lateral seta present, segment apically elongate B lateral seta present, segment not apically elongate, short C lateral seta absent, segment apically elongate D lateral seta present, segment elongate, with additional segmental processes, branched or bifurcate E lateral seta absent, segment elongate, with outer segmental processes F lateral seta absent, segment short, apically truncate or blunt, leg rami symmetrical or asymmetrical.
terns are those lacking a lateral seta (i.e., E, C, and F). The known distribution of records of these six groups is presented in Fig. 5. Group D is restricted to the Mediterranean and Japan; the primitive patterns A and B are the most widespread, distributed in the most diverse regions (i.e., the Mediterranean, northeastern Atlantic) and reaching polar and subpolar latitudes. The derived groups C and F occur in the most diverse regions but they are the only groups present in the Western Hemisphere, restricted to the NWTA region. The distribution of the species diversity and our interpretation of the female fifth leg types suggest the occurrence of different colonization and speciation events in these coastal demersal copepods resulting from geological changes (i.e., marine regressions and transgressions) in each region (see Fleminger 1986;Por 1986;Suárez-Morales 2003). The genus probably passed through different episodes of diversification mainly in regions such as the western Pacific and the Mediterranean, where both primitive and derived patterns of the female fifth legs co-occur (Fig. 5). The complex biogeographic history of the Mediterranean explains the high diversity and co-occurrence of species belonging to at least three of our groups, suggesting distinct origins, and remarkably characterized by their preference for anchialine and cave habitats (Por 1986;Jaume et al. 2008;Kršinić 2015). Contrastingly, the most recent radiation of Stephos appears to have taken place in the NWTA, represented by only four species with a derived female fifth leg; three of them are anchialine (S. lucayensis, S. exumensis, S. fernandoi) (Fosshagen 1970;present data) and one is planktonic (S. deichmannae) (Fleminger 1957). Furthermore, the new species, S. fernandoi, has clear affinities with the two species of Miostephos, mainly in the reduced female fifth leg (as in pattern C) (Bowman 1976;Yeatman 1980); it is thus suggested that Stephos-like benthopelagic ancestors invaded the region, colonized the caves and one branch subsequently diverged into the Miostephos lineage in the NWTA. Similar colonization processes, with diversity/radiation centers in the western Pacific and congeners distributed in the NWTA have been described for other demersal calanoids like Tortanus (Ohtsuka & Reid, 1998) and Bestiolina (Suárez-Morales & Almeyda-Artigas, 2016). It is speculated that ancestors of Stephos probably originated in the Australian-Western-Pacific region and successively colonized the Mediterranean and the northeastern Atlantic. They probably reached the NWTA either through the Isthmus of Panama during the Middle Miocene-Pliocene (Ohtsuka and Reid 1998) although there are no records of Stephos from the eastern Pacific coast, or by passive transportation of planktonic forms onto the NWTA. This analysis should be considered tentative as there are still many missing data; a complete morphological revision of incompletely described species, a confirmation of doubtful records, and a phylogenetic analysis including molecular data is expected to reveal more detailed patterns about this genus.