A new species of Metacyclops Kiefer, 1927 (Copepoda, Cyclopidae, Cyclopinae) from the Chihuahuan desert, northern Mexico

Abstract A new species of the freshwater cyclopoid copepod genus Metacyclops Kiefer, 1927 is described from a single pond in northern Mexico, within the binational area known as the Chihuahuan Desert. This species belongs to a group of Metacyclops species with a 3443 spine formula of swimming legs. It is morphologically similar to Metacyclops lusitanus Lindberg, 1961 but differs from this and other congeners by having a unique combination of characters, including a caudal rami length/width proportion of 3.5–3.8, a innermost terminal seta slightly longer than the outermost terminal seta, intercoxal sclerites of legs 1-4 naked, a strong apical spine of the second endopodal segment of leg 1 and one row of 6-8 small spinules at the insertion of this spine. The finding of this species represents also the first record of the genus in Mexico and the third in North America, where only two other species, Metacyclops gracilis (Lilljeborg, 1853)and Metacyclops cushae Reid, 1991 have been hitherto reported. This is also the first continental record of a species of Metacyclops from an arid environment in the Americas. This species appears to be endemic to the Chihuahuan Desert, thus emphasizing the high endemicity of this area.

leg, along with many other currently neglected characters will result in splitting the genus Metacyclops into several different genera. The recent work by Karanovic et al. (2011) included a cladistic analysis and a taxonomic revision of the Australian species. These authors formally recognized the "Metacyclops complex" and provided evidence to state that Metacyclops s. str. is polyphyletic in nature and that some species (like M. cushae) are closer to other genus than they are to Metacyclops.
Most of the surveys on the Mexican cyclopoid copepod fauna have dealt with the central and southern regions (Suárez-Morales and Reid 1998, Suárez-Morales et al. 2002. Recent research efforts in arid and semi-arid regions of Central and Northern Mexico (Dodson and Silva-Briano 1996, Mercado-Salas et al. 2006, Suárez-Morales and Walsh 2009Suárez-Morales 2009, 2011) indicate that the copepod fauna of these habitats is more diverse than previously thought and that it deserves further study. During a recent revision of zooplankton collections from arid areas of Northern Mexico, several specimens of cyclopoid copepods were identified as representing an undescribed species of Metacyclops. The significance of this finding is discussed in terms of currently known diversity and distributional patterns of the genus in the Americas.

Methods
During the development of a project to estimate the diversity of cyclopoid copepods in arid and semi-arid regions of Mexico, zooplankton samples were collected between 1981 and 2009 in more than 500 water bodies from six states of Central and Northern Mexico (Aguascalientes, Chihuahua, Coahuila, Durango, San Luis Potosí, and Zacatecas). Samples were collected using a conical standard plankton net (250 mm diameter and 50 µm-mesh size) hauled near the shoreline of water bodies. The biological material was then fixed and preserved in 4% formalin solution. Copepods were sorted out from the original samples and then transferred to 70% ethanol with a drop of glycerine for long term preservation. Several female and male specimens of a cyclopoid copepod were collected from Coahuila in northern Mexico. These copepods were tentatively identified as Apocyclops panamensis (March, 1913). A second, closer examination of these specimens was performed in the laboratory and differences with respect to A. panamensis motivated a deeper analysis which revealed these specimens as members of Metacyclops. The taxonomically relevant characters of this genus were evaluated following Reid (1987Reid ( , 1991, Herbst (1988), Rocha (1994), Karanovic (2004aKaranovic ( , 2004b, and Karanovic et al. (2011). Specimens were processed for taxonomical examination following Reid and Williamson (2010). Three females were dissected and 10 females were prepared for SEM examination with a JEOL LV 5900 microscope at facilities of the Universidad Autónoma de Aguascalientes, Mexico. The SEM processing included dehydration in progressively higher ethanol concentrations (60, 70, 80, 96, 100%), drying, and gold coating following standard methods. All dissected specimens were mounted in semi-permanent slides with glycerine sealed with Entellan®, a commercial, fast drying mounting medium and sealant. Scaled illustrations were done at 100X magnifications with a drawing tube mounted on a standard Olympus CX31 microscope. Type specimens were deposited in the collection of zooplankton held at El Colegio de la Frontera Sur, in Chetumal, Mexico (ECO-CH-Z) and in the Laboratorio de Ecología of the Universidad Autónoma de Aguascalientes, Mexico.  specimens,undissected,. Original plankton samples containing several additional specimens, and the SEM-processed specimens are deposited in the collection of M. Silva-Briano, Laboratorio de Ecología of the Universidad Autónoma de Aguascalientes, Mexico. Samples from the type locality were collected by Alejandro Maeda-Martínez in October 10, 1981.

Order
Type locality. Ephemeral pond at El Refugio bridge, Cerro Bola, Km 70, east of Torreón city, federal highway 40, Coahuila (25°35'02"N, 102°45'02"W). This pond is located in a desertic plain in the southwest margin of the ancient Laguna de Mayrán, a system which is part of the endorheic drainage of the Nazas and Parras rivers. The altitude of the type locality is about 1,100 meters above sea level and the average annual precipitation in the area is 200 mm. At the moment of sampling the surface area of the pond was about 55 meters long and 25 meters wide, the water had an average depth of 50 cm and 80 cm at its deepest point.
Etymology. The specific epithet makes reference to the arid habitat from which this species was collected. It was used to emphasize that it is the first American record from arid conditions.
Descriptions. Female: Habitus as in Fig. 1A (dorsal view) and Fig. 4A (lateral view). Length of holotype 0.87 mm from anterior end of cephalothorax to posterior margin of caudal rami (range=0.72-0.87 mm; mean=0.80 mm; n=9). Body robust, cephalothorax relatively long, slightly expanded laterally at midlength of cephalosome in dorsal view; lateral margins of pedigers 3 and 4 straight, produced posteriorly. Cephalothorax length= 0.55 mm, representing 63% of total body length. Dorsal surface smooth, antennules not reaching distal margin of first pediger. Urosome (excluding caudal ramus) (Fig. 1B) representing 37% of body. Posterior margins of genital double-somite, free urosomites, and anal somite smooth both dorsally and ventrally. Relative length of each urosomite (proximal to distal) as: 65.4: 10.3: 10.3: 14.1=100. Genital double-somite (Fig. 5E) representing 17% of body length (excluding caudal rami), somite about 1.1 times longer than broad, with maximum width at proximal half; ventral and dorsal surfaces smooth. Anterior half of genital double-somite expanded laterally. Seminal receptacle with a reduced and narrow anterior part, posterior part rounded and expanding along the somite. Anal somite with distal rows of spines at insertion points of each caudal rami on ventral and dorsal margins. Anal operculum ( Fig. 1I) slightly rounded and smooth.
Antenna (Fig. 1D, 4C): Four-segmented, basis without cuticular ornamentation, armed with long exopodal seta and two basipodal setae of different size, outer seta 1.6 times longer than inner seta. First endopodal segment with single outer seta and inner group of spinules. Second segment with 6 setae; inner margin with longitudinal row of spinules. Third endopodal segment with 6 terminal setae; inner margin with row of spinules.
Mandible (Fig. 1E): Gnathobase with 7 strongly chitinized teeth and dorsal seta armed with inner row of spinules. Palp reduced, with 2 long and 1 short setae, the later not reaching half-length of former two.
Maxilliped (Figs. 1H, 4D): Four-segmented. Syncoxa with 3 spiniform setae along inner margin: proximal one without ornamentation at insertion, middle one longest, more than twice as long as the other setae. Basis with 2 spiniform setae and transverse row of spines. Endopod reduced, 2-segmented, first segment with single lightly spinulate seta. Second endopodal segment armed with spiniform proximal seta and 2 slender setae.
Legs P1-P4: with naked intercoxal sclerites, distal margins with rounded projections. All endopodal and exopodal setae slender and plumose. Armature formula of all swimming legs as in Table 1.
Leg 1 (Fig. 2A, 5A): Coxa with inner seta and transverse row of 6 spinules on distal outer margin. Basis with inner row of short setae and long slender basipodal seta, reaching middle margin of second endopodal segment, row of hair-like setules along inner margin, row of 5 spines adjacent to insertion of endopodal ramus. Endopod slightly shorter than exopodite. Apical spine of second endopodal segment strong, slightly longer than segment, with spinules at insertion point.
Leg 2 (Fig. 2B): Coxa with inner seta . Basis with short slender seta on outer margin. Surface of coxa and basis smooth. Endopod slightly shorter than exopodite.
Leg 4 (Figs. 2D, 5B): Coxa and basis as in legs 2-3. Endopod shorter than exopodite. Second endopod about two times longer than wide (1.9), with apical spine shorter than bearing segment(0.8 times as long as segment). Spinules at insertion of all elements of second endopodal segment. Second exopodal segment with 2 outer spines and 1 apical spine with small spinules at insertion point.
Leg 5 (Figs. 1B, 2E, 5C): Basal segment completely fused to somite, dorsal seta stout and plumose, about 1.4 times longer than outer seta of free segment. Free segment subrectangular, 1.2 times longer than wide, inner spine slightly shorter than bearing segment. Outer seta about 4 times longer than inner spine. Inner spine strong and smooth; outer seta plumose on distal half.
Leg 6 (Figs 1B, 5D): Represented by small, low plate near lateral margin of genital double somite. Leg armed with relatively long plumose seta, and with 2 short, subequal smooth spines.

Antenna, mouthparts and legs 1-3 as in female.
Leg 4 (Fig. 3C): as in female except for relatively longer exopodite. Leg 5 (Fig. 3E): Basal segment completely fused to somite, dorsal seta stout, as long as outer seta of free segment. Free segment subrectangular, 1.5 times longer than wide, spine as long as segment, outer seta about 5 times longer than inner spine. Inner spine strong, smooth; outer seta plumose on distal half.
Leg 6 ( Fig. 3F): Represented by small, low plate near lateral margin of genital somite with relatively strong and long inner spine, two outer setae about the half of length of inner spine. Spine and setae smooth.
Remarks. The only two other species of the genus known to occur in North America, M. cushae Reid, 1991 and M. gracilis (Lilljeborg, 1853), are easily distinguishable from the new species. The former species belongs to the "Group C" (Karanovic 2004a(Karanovic , 2004b, with a 3433 spinal formula, being the only species in the group. Metacyclops gracilis belongs, like the new species, to Karanovic´s (2004a, 2004b) "Group A". The new species differs from M. gracilis mainly by its having of one apical spine on the second endopodal segment of leg 4, instead of two such spines present in M. gracilis.
Following the comprehensive key to the known species of Metacyclops (Herbst 1988), the new species was tentatively identified as M. lusitanus Lindberg, 1961 from Portugal because both share several characters including: 1) 11-segmented female antennules, 2) margins of all somites smooth, 3) one spine on the apical margin of the second endopodal segment of leg 4, 4) inner apical seta of caudal ramus shorter than the outer seta, 5) length/width proportion of caudal ramus (about. 4.0), 6) apical seta of fifth leg 4 times longer than the apical spine, and 7) apical spine of the second endopodal segment of leg 4 shorter than the segment. However, a closer examination showed several differences between these two species. In M. lusitanus the dorsal caudal seta (VII)/outermost terminal seta (III) length ratio (0.7) differs from that found in the new species (0.8). In addition, the length ratio of the outermost terminal seta(III)/ innermost terminal seta (VI) of the caudal ramus differs between these species, in M. lusitanus it is 1.7 vs. 1.1 in the Mexican species. The length ratio of the basipodal seta/ total length of endopod of leg 1 slightly differs in these species, this ratio being 0.9 in the M. lusitanus and 0.85 in the new species. Also, the apical spine of the second endopodal segment of leg 1 is clearly stronger in M. deserticus sp. n. than it is in M. lusitanus (see Lindberg, 1961, fig. 1b). Metacyclops deserticus sp. n. has 6-8 small spinules at the insertion of the apical spine whereas such ornamentation is absent in M. lusitanus. We also followed Reid's (1987) key to the American species. Our specimens key down to M. curtispinosus Dussart, 1984. The new species shares several characters with M. curtispinosus, including 11-segmented antennules, also present in M. agnitus Herbst, 1988, M. pectiniatus Shen and Tai, 1964, M. subdolus Pesce, 1978, M. hannensis Defaye, 1992, and M. gasparoi Stoch, 1987. All of them belong to Karanovic´s (2004aKaranovic´s ( , 2004b (1.2-1.3). An additional difference between these species is the shape of the inner margin of the leg 4 basis. In the new species but also in M. curtispinosus, M. agnitus, M. pectiniatus, and M. hannensis it is rounded vs. triangular-in M. subdolus and M. gasparoi.
In addition, the new species differs from its congeners in the length of the external seta of free segment of P5/ inner spine length ratio; in the new species, this ratio is 4.0, whereas it ranges between 2.9 and 3.1 in M. subdolus and between 5.0 and 5.7 in M. hannensis, M. pectiniatus and M. agnitus. In M. gasparoi this value is 6.6 and in M. curtispinosus it is about 8.0. The proportion between inner spine/length of segment of P5 is a character that also differs among these species. In M. curtispinosus the ratio is 0.3, in the new species it is about 0.8, in M. hannensis and M. agnitus the spine is as long as the segment, in M. pectiniatus and M. gasparoi it is about 1.3, in M. subdolus 1.5 times. Also the length proportion of the seta of fifth leg fused to the segment/outer seta of free segment represents a character that differs between species, in M. gasparoi it is 0.6, M. hannensis and M. subdolus have a proportion ranging between 0.8 and 0.9. M. agnitus and M. curtispinosus shares similar values (1.1-1.2) and both M. pectiniatus and the new species have a length ratio close to 1.4 (Herbst 1988, Pesce 1985, Lim and Fernando 1985, Pesce 1978, Defaye 1992, Stoch, 1987. The length/width ratio of the caudal ramus also differs among these species, M. curtispinosus has relatively low value (2.4-2.8) that differs from those in M. agnitus, M. pectiniatus, and M. hannensis (3.2-3.4). Metacyclops subdolus has a wide range of variation (2.9-3.4). The new species has a relatively longer caudal ramus (3.5-3.8), but it is shorter than in M. gasparoi (5.5-5.7). Another valuable character is the length ratio of innermost terminal seta/outermost terminal seta; we found two main groups for this character. In the first one the innermost terminal seta is shorter than the outermost terminal seta; this character is present in M. agnitus (0.5), M. pectiniatus (0.6), M. hannensis (0.7), and in the new species (0.8). In the second group the innermost terminal seta is longer than outermost terminal seta: M. curtispinosus (1.2), M. subdolus (1.5-1.7), and M. gasparoi (2.0). In addition, the length dorsal seta/length of caudal ramus ratio also separates two groups. In the first group, the dorsal seta is shorter than the ramus: M. deserticus sp. n. (0.4-0.5), M. curtispinosus and M. hannensis (0.7). In the second group the dorsal seta is longer than caudal ramus like in M. gasparoi, M. agnitus (1.1), and M. subdolus (1.8-2.1).

Discussion
Metacyclops deserticus sp. n. from northern central Mexico represents the first new species of the genus Metacyclops described in this country and is also the third record for North America (Reid 1991, Reid andWilliamson 2010). There is a previous record of M. cushae by Gutiérrez-Aguirre and Cervantes-Martínez (2013) from southeast Mexico (Chiapas State). Most of the known American species of Metacyclops occur in tropical environments of the Neotropical region, mainly in South America and only M. cushae and M. gracilis have been reported from the Neartic region (Dussart and Defaye 2006). All previous records of species of Metacyclops in the Americas are from tropical environments; the North American records are from marshes in Louisiana and the Everglades in Florida (Bruno et al. 2005, Reid 1991). The finding of this species from an arid environment in Mexico represents the first continental record of the genus from this kind of habitat Defaye 2006, Rocha 1994). Members of Metacyclops have been recorded from arid environments in other regions of the world like Australia, New Zealand and Africa; some of these species inhabit epigean systems but others are known from subterranean waters (Dussart 1977, Dumont 1981, Defaye 1992, Pesce et al. 1996, Karanovic 2004a, 2004b. According to Defaye (1992) species of Metacyclops usually inhabit small pools, wells and ponds rich in vegetation and organic material, and their presumed tolerance to high temperatures and salinities appear to predict a wider distribution in Africa and other places with arid conditions, now including the American desert systems.
The arid areas of north-central Mexico were formed between the Late Oligocene and Middle Miocene (30-20 MYA), and were part of a general trend toward a greater aridity resulting from climate changes associated to the intense volcanic activity and tectonics that characterized the Cenozoic. The Rocky Mountains, the Mexican and Central-American Plateaus, and the sierras Madre were formed as result of tectonic activity during Cenozoic. The formation of the Sierra Madre Occidental and Sierra Madre Oriental during the Eocene and continuing until the middle Miocene provided a new barrier to the atmospheric flow. This barrier blocked the masses of warm, moist air from the Pacific Ocean and the Gulf of Mexico and caused a severe drought and desertification of the Mexican Plateau. The Mexican Plateau includes the states of Coahuila, Chihuahua, Zacatecas, Durango. The Miocene climate change segregated the species along latitudinal and longitudinal gradients, thus favoring radiation processes of some lineages (Devitt 2006). It has been suggested that some of these areas of Northern Mexico functioned as refugia during the Pleistocene glaciations, thus favoring local process of isolation-speciation of the aquatic biota (Bănărescu 1991, Suárez-Morales et al. 2010, Mercado-Salas et al. 2012. The cyclopoid copepod fauna of arid areas of central-north of Mexico (Chihuahuan Desert) is currently represented by 39 species belonging to 12 genera. This binational zone is currently deemed as an area with a high endemicity; up to 20% of these species are endemic to these arid areas (Mercado-Salas et al. 2006, Suárez-Morales and Walsh 2009, Suárez-Morales et al. 2010. The new species of Metacyclops appears to be endemic to the Chihuahuan Desert, thus incrementing the importance of this and other arid systems as refuges of an undescribed diversity that certainly deserves further study. Most interestingly, the type locality harbors at least three endemic crustacean species, the copepod M. deserticus and the large branchiopods anostraceans (fairy shrimps) Branchinecta oterosanvicentei Obregón-Barboza, Maeda-Martínez, García-Velazco and Dumont, 2002, andStreptocephalus guzmani Maeda-Martínez, Belk, Obregón-Barboza andDumont, 1995.