Two new species of Monstrilla (Copepoda: Monstrilloida: Monstrillidae) from a protected reef system of the Mexican Caribbean

. Several adult female monstrilloid copepods, collected in March 2022 from the protected reef area of Xcalak, on the southern part of the Mexican Caribbean coast, proved to belong to two undescribed species of Monstrilla Dana, 1849. They are described here as M. xcalakensis sp. nov. and M . annulata sp. nov., partly by use of scanning electron microscopy. Females of the two species are generally similar but differ in: (1) antennular segmental structure, (2) antennular armature and ornamentation, (3) structure and setation of the ﬁ fth leg, (4) number and modi ﬁ cations of the caudal setae, and (5) integumental ornamentation. Comparison with congeneric species revealed distinctive features for both species that support their status as new. These two species are interesting additions to the reef-dwelling monstrilloid copepod fauna of the Mexican Caribbean and con ﬁ rm Monstrilla as the most diverse genus of monstrilloids in this area, now represented by nine species.


Material and methods
Several female monstrilloids referable to the genus Monstrilla were isolated from light-trap samples collected in March 2022 from Xcalak, a protected reef system on the southern part of the Mexican Caribbean coast (approx.18°16′ N, 87°50′ W).Detailed microscopic examination showed them to belong to two undescribed species of the genus, which are described here as much as possible by following the criteria proposed by Grygier & Ohtsuka (1995, 2008) for female monstrilloids.The descriptions of both species are partly based on SEM observations that provide insight into interesting morphological details.Two adult females of each species were prepared for SEM examination by dehydration of specimens in progressively stronger aqueous solutions of ethanol (70-100%), criticalpoint drying, and gold-coating following standard procedures.Observations were made with a JSM-6010LA microscope at El Colegio de la Frontera Sur, Chetumal, Mexico.The type specimens have been deposited in the Zooplankton collection (ECO-CHZ) at El Colegio de la Frontera Sur (ECOSUR) in Chetumal, Mexico.The new species were compared with their known congeners based on published literature.

Results
Phylum Arthropoda von Siebold, 1848Subphylum Crustacea Brünnich, 1772Superclass Multicrustacea Regier et al., 2010Subclass Copepoda Milne Edwards, 1840Order Monstrilloida Sars, 1901Family Monstrillidae Dana, 1849 Genus Monstrilla Dana, 1849 The two species studied here can be assigned to the genus Monstrilla by the presence of 1) a single urosomite between the genital double-somite and the anal somite, 2) well-developed fi fth legs, with two ramal lobes usually carrying setae, and 3) six setae on each caudal ramus.This combination of characters is unique among the accepted genera of monstrilloids (Isaac 1975;Suárez-Morales & McKinnon 2014;Jeon et al. 2018).

Differential diagnosis
Medium-to large-sized female Monstrilla with elongate, cylindrical cephalothorax constituting more than 60% of body length and prominent oral cone situated one-third of way along ventral surface of cephalothorax.Antennule four-segmented and segments 1-4 separate.Outer exopodal spines of legs 1-4 long and acute.Genital double-somite with anterioventral expansion carrying pair of posteriorly directed ovigerous spines, these not reaching to tips of caudal rami.Fifth legs bilobed, both lobes of similar length and breadth (endopodal lobe slightly shorter) and fused to each other in distal half; endopodal lobe armed with single seta, exopodal lobe with three setae.Caudal rami each bearing six setae, with proximal ends of apical setae III and IV much swollen.

Etymology
The specifi c name, an adjectival Mayan toponym (+ Latin suffi x '-ensis' = 'place'), refers to the reef system of Xcalak in the Mexican Caribbean, where this species was collected.The correct pronunciation of the species name starts with 'sh', followed by a strong 'ka'.Gender is feminine.
Other material MEXICO • 1 ♀, undissected, glycerin-preserved in vial; same collection data as for holotype; author's collection at ECOSUR.
CEPHALOTHORAX.Long, cylindrical, with straight margins, representing about 60% of total body length and fully incorporating fi rst pedigerous somite.Oral cone prominent (oc in Fig. 3B), located 29% of way back along ventral surface of cephalothorax (Figs 1B,3B).Cephalic region anteriorly subquadrate in dorsal view, 'forehead' fl at with pair of small sensilla (arrowheads in Fig. 1A).Lateral cups of eye poorly developed, almost unpigmented (lc in Fig. 3B).Ventral cup relatively small (vcu in Fig. 3B), of nearly same diameter as lateral cups.Cuticular ornamentation of 'facial' region of cephalothorax consisting of  fi elds of longitudinal and oblique striae between oral cone and bases of antennules (Figs 1D, 3B); two clusters of three protruding pores each, these being located on anterioventral medial surface between antennule bases (Fig. 1D: white arrowheads); two pairs of nipple-like processes lateral and anteriolateral to oral cone, each with pore at apex, with additional indistinct pair between and slightly mesial to these (Fig 1D ); and two pairs of minute pores at anteriolateral base of oral cone (Fig. 1D).

Differential diagnosis
Medium-to large-sized female Monstrilla with long cephalothorax.Antennule 4-segmented, with reduced setal armature: segment 1 unmodifi ed; segment 2 lacking all elements of '2v' group (i.e., 2v 1-3 ); segment 3 with usual armature and 3 weak integumental constrictions; and segment 4 with 5-7 deep integumental constrictions making it appear multi-annulate, with apex modifi ed into diagonally truncate tip forming fl at, disc-like structure with reduced armature, i.e., lacking usual apical elements 6 1 and 6 2 but retaining reduced 6aes.Fifth legs weakly bilobed, with endopodal lobe represented by short, fl at protuberance on proximal inner margin of exopodal lobe, latter represented by large, subrectangular plate with incised inner margin, distinctively pilose outer margin, and one distal and one subdistal seta.Ovigerous spines not reaching to distal margins of caudal rami.Surface of caudal rami and other appendages, including proximal parts of ovigerous spines and urosomites (Fig. 6E), densely sculptured with fi ne, thread-like undulate patterns.Caudal rami each bearing fi ve setae.

Etymology
The specifi c name is a Latin adjective with a feminine suffi x to match the gender of the genus, meaning 'ringed' in reference to the worm-like appearance of the integumental constrictions of antennular segments 3 and 4.
Other material MEXICO • 2 ♀♀, undissected in vial with glycerol; same collection data as for holotype; author's collection at ECOSUR.
CEPHALOTHORAX.Relatively robust, representing about 63% of total body length and fully incorporating fi rst pedigerous somite.Oral cone prominent, located 30% of way back along ventral surface of cephalothorax.Cephalic region anteriorly subquadrate in dorsal view, 'forehead' fl at, sensilla not observed.Nauplius eye as in M. xcalakensis sp.nov.except for lateral cups being almost unpigmented.Cuticular ornamentation of cephalothorax including medial cluster of minute, crater-like pores between antennule bases (Fig. 5A), two pairs of nipple-like processes between oral cone and antennule bases, and fi eld of transverse striae adjacent to oral cone (Fig. 6C).

Justifi cation for establishment of M. annulata sp. nov.
The new species Monstrilla annulata sp.nov.has two distinctive features that make it readily distinguishable among its congeners and support its status as a new member of the genus Monstrilla.First is the modifi ed antennule with integumental constrictions appearing as rings, particularly in segments 3 and 4. A generally similar pattern is known in Spinomonstrilla spinosa Suárez-Morales, 2019, but in that species the annulated appearance is imparted by arcuate protruding scales, not by simple circular constrictions (cf.Suárez-Morales 2019: fi g. 2d-e).Also, the antennules are remarkably elongate and narrow in S. spinosa, with the setal groups of segments 2-4 being more widely separated from each other than in M. annulata.
The second distinctive feature of M. annulata sp.nov. is the uniquely modifi ed exopodal lobe of the female's fi fth leg; no other member of Monstrilla has a fi fth leg combining an indented inner margin and a heavily hirsute outer margin.A lightly hirsute outer margin is known only in the female Caromiobenella brasiliensis (Suárez-Morales & Dias 2000), in which, however, the exopodal lobe carries three seta and the endopodal lobe is cylindrical and narrow (cf.Suárez-Morales & Dias 2000: fi g. 3d), thus differing from M. annulata.A spinulose outer margin of the fi fth leg is present in the poorly described M. dakinensis Davis, 1949 from Australia (Dakin & Colefax 1940), which shares with M. annulata the same armature of two setae on the exopodal lobe and a reduced endopodal lobe, but differs in lacking the indentations in the exopodal lobe's inner margin that help to defi ne the new species.An exopodal lobe with two setae is also known in the Caribbean M. elongata, but the lobe is bulbous, not plate-like, and the antennules are unsegmented (cf.Suárez-Morales 1994: fi g. 1c-d, g), unlike in the new species M. annulata.

Discussion
No other species of Monstrilla besides the new species M. xcalakensis sp.nov.and M. ilhoii has a fi fth leg showing distal fusion of the endopodal and exopodal lobes.The developmental stages of monstrilloids have been studied in some detail by Huys et al. (2007), focusing on the antennules and caudal rami of males, but the possible ontogenetic origin of ramal fusion in the female's fi fth leg requires closer attention.According to Huys & Boxshall (1991), this leg is biramous, both rami are fused to the protopodal part of the limb, and the protopods of the left and right members of the leg pair are not connected by an intercoxal sclerite.An account of the development of the female fi fth leg from the CIII stage to the preadult CV in an unidentifi ed species of Monstrilla (Suárez-Morales et al. 2014) revealed that at the CIII stage, the fi fth leg is represented by a single lobe, which progressively splits into a pair of distal lobes.Before this, the fi fth legs were very likely a pair of unarmed buds.Completion of the fi nal setation pattern is attained at the CIV stage, while separation of the lobes is still in progress.The endopodal lobe then becomes clearly separated from the exopodal lobe by the time the copepod exits its host as a preadult CV.It seems more likely that the unusual fi fth legs of M. xcalakensis and M. ilhoii are the result of incomplete separation of the lobes in ontogeny, not a secondary but incomplete re-fusion of fully-formed lobes during the copepodite phase of development.The juvenile development of both species needs to be studied and compared to confi rm this.
The structure of crustacean setae is highly variable, being defi ned by their function.In Garm & Watling's (2013) categorization of the known types of arthropod setae, none displays the type of grooved structure found in some of M. annulata's caudal setae.Since such grooves do not occur in any recognized setal type, their function and origin remain speculative.It is also possible that the grooves are shrinkage artifacts resulting from the dehydration and critical point drying procedures employed prior to SEM observation.Caudal setae of other specimens of the type series examined by light microscopy had a row of setule scars but no obvious grooves.
The student who initially sorted the samples from Xcalak assumed that the female specimens of Monstrilla xcalakensis sp.nov.and M. annulata sp.nov.belonged to a single species, as they generally resemble each other.Superfi cial examination of plankton samples, particularly in highly diverse habitats for monstrilloids like coral reefs (Sale et al. 1986;Suárez-Morales 2001, 2003;Grygier & Ohtsuka 2008), can fail to distinguish among morphologically similar species.As many as eight species of monstrilloids had been described or reported from different reef zones of the Mexican Caribbean coast before the present study.This coast, which is part of the Mesoamerican Barrier Reef System (MBRS), supports a wide variety of aquatic habitats, including northern (Puerto Morelos = PM) and southern (Mahahual-Xcalak = MXK) reef sectors and Banco Chinchorro (BCH), an oceanic atoll.The species reported from these areas are M. mariaeugeniae (PM), M. elongata (PM, BCH), M. mahahualensis (PM, MXK), M. xcalakensis (MXK), M. annulata (MXK), M. careli (BCH), M. careloides (BCH), M. marioi (BCH), M. globosa (BCH), and Cymbasoma quintanarooense Suárez-Morales, 1994 (BCH) (Suárez-Morales 1994, 1996, 2003, 2022b).Monstrilla is clearly the most diverse genus of monstrilloid copepods in Mexican Caribbean reef areas, thus providing a contrast with other coastal and estuarine habitats like those in Brazilian waters, where the monstrilloid diversity is relatively low and mainly confi ned to coastal, transitional, and estuarine areas (Suárez-Morales & Dias in prep.), or in the southern Gulf of Mexico and northern coast of the Yucatan Peninsula, where only three species, M. humesi, C. quintanarooense, and C. chelemense Suárez-Morales & Escamilla, 1997, have been reported (Suárez-Morales 1993;Suárez-Morales & Escamilla 1997, 2001), all from shallow, low-salinity coastal systems.The diversity of monstrilloids has been underestimated in western Caribbean marine habitats, and the list is expected to grow as new sampling programs are undertaken.
The males of both new species described herein remain unknown.One of the main problems in monstrilloid taxonomy has been to reliably match both sexes of a species (Suárez-Morales 2010, 2011, 2018;Jeon et al. 2018b), a task that is particularly diffi cult in highly diverse habitats like coral reefs.The Mexican Caribbean reef system harbors at least three species of Monstrilla with males that have a genital complex like that of M. conjunctiva Giesbrecht, 1893 (see Suárez-Morales 2022b) and craterlike processes on antennular segments 2-4 (Suárez-Morales 1996, 1998, 2022).Members of this group are now known to be distributed in three different reef areas of the Mexican Caribbean, PM, MXK (M.mahahualensis), and BCH (M.elongata), but females are known only for M. elongata.It is possible that one or both of the present new species, currently known only from females, will eventually be matched by means of life-history studies or molecular systematics to one or the other of this region's current male-only species.