A new species of Mastigodiaptomus Light, 1939 from Mexico, with notes of species diversity of the genus (Copepoda, Calanoida, Diaptomidae)

Abstract A new species of the genus Mastigodiaptomus Light, 1939, named Mastigodiaptomus cuneatus sp. n. was found in a freshwater system in the City of Mazatlán, in the northern region of Mexico. Morphologically, the females of this new species are distinguishable from those of its congeners by the following combination of features: the right distal corner of the genital double-somite and second urosomite have a wedge-shaped projection, the fourth urosomite has no dorsal projection and its integument is smooth. The males are distinct by the following features: the right caudal ramus has a wedge-shaped structure at the disto-ventral inner corner; the basis of the right fifth leg has one triangular and one rounded projection at the distal and proximal margins, respectively, plus one hyaline membrane on the caudal surface close to the inner margin; the aculeus length is almost the width of the right second exopod (Exp2); and the frontal and caudal surfaces of the right Exp2 are smooth. Furthermore, the analysis of the COI gene of Mastigodiaptomus cuneatus sp. n. has revealed that Mastigodiaptomus albuquerquensis (Herrick, 1895) is its nearest congener, with 18.64% of genetic distance. A key for the identification of the known species of the genus is provided.

The fine structural features of the anatomy of the females and males of freshwater copepods are informative for species recognition. Researchers that have developed this idea concerning free-living copepods are Van de Velde (1984) and Hołyńska (2000) in the Cyclopidae genus Mesocyclops; and Alekseev et al. (2006) in the Cyclopidae genus Eucyclops, particularly the Eucyclops serrulatus species complex. Bowman (1986) recognized morphologically similar species within the Mastigodiaptomus genus using differences in the integument of prosomal wings and fifth legs of both sexes. Empirical evidence gathered from several species of freshwater crustaceans has shown that these morphological differences are consistent with reproductive isolation (Alekseev et al. 2006), genetic differentiation (Quiroz-Vázquez and Elías-Gutiérrez 2009; Gutiérrez-Aguirre et al. 2014), or both when they are probed (Montiel-Martínez et al. 2008).
In the present work is provided an illustrated description of both sexes of one new species of the genus Mastigodiaptomus; it was found in the northern region of Mexico in a field collection carried out in 2014. The analysis was based upon the detailed micro-structure of the antennules, fifth legs, and prosomal integument. In addition, the sequences of the mitochondrial COI gene were used to assess the genetic divergence between the new species and seven congeners to incorporate molecular information into the species description.

Methods
Morphological analysis. The cephalic appendages, swimming legs and urosome of Mastigodiaptomus cuneatus sp. n. were examined using light microscopy and illustrated with the aid of a camera lucida. The specimens were dissected and appendages were mounted in glycerine.
The terminology and abbreviations used for the armament of each appendage and structure are based on Huys and Boxshall (1991): spiniform process ae aesthetasc Enp1-Enp n first to "n" endopodal segment Exp1-Exp n first to "n" exopodal segment P1-P5 Legs 1-5 The type material was deposited at the Colección de Referencia de El Colegio de la Frontera Sur (ECOCH-CHZ) Chetumal, México and the Colección Nacional de Crustáceos (CNCR) del Instituto de Biología, Universidad Nacional Autónoma de México.
Molecular analysis. Specimens (2 females, 3 males, and 3 copepodites) were preserved after capture in 96% ethanol and prepared for barcoding following standard methods. DNA was extracted using the HOTSHOT method (Montero-Pau et al. 2008). A segment of the COI gene was amplified using the LCOI490 and HCO2198 primers or the Zplank primers, as suggested by Folmer et al. (1994) and Prosser et al. (2013), respectively. The preparation of the PCR 12.5 µL PCR reaction mixture and visualization of PCR products was performed as described by Gutiérrez-Aguirre et al. (2014) and Montiel-Martínez et al. (2008). Sequence analysis was carried out at the Chetumal Node of the MEXBOL (El Colegio de la Frontera Sur).
Additionally, a search of GenBank and the public data of the Barcode of Life Data System (BOLD) produced sequences of Mastigodiaptomus albuquerquensis, M. patzcuarensis, M. cf. albuquerquensis (accession numbers for BOLD and GenBank in Gutiérrez-Aguirre et al. 2014), M. texensis, M. cf. nesus, M. montezumae, M. cf. reidae, and M. reidae (accession numbers for BOLD and sampled site are shown in Table 1).
These sequences were downloaded from the BOLD project files Mastigodiaptomus of Mexico (MALB), Microcrustacean from Mexico (MCM), and Zooplankton II (ZPII) and compared with our sequence of M. cuneatus sp. n., this latter sequence is into the project MCM. In these project files, the electropherograms, sequence data, photographs, primers data, and collection details are available (on the Barcode of Life Data System http://www.boldsystems.org). Fifty-two COI gene sequences > 500 bp were used for the analysis, and BOLD Aligner and the ID Tree using the model Kimura 2 parameter (K2P; Kimura 1980) were utilized to obtain the ID Tree. Two sequences of Hesperodiaptomus arcticus (Marsh, 1920) (Calanoida: Diaptomidae) were used as an outgroup.  Type locality. A lagoon called Laguna El Camarón in Avenida Insurgentes, Mazatlán, Sinaloa City, México; 23°14'10"N; 106°26'18"W.
Etymology. The name of the species means "wedged" in Latin and refers to the chitinous protuberance present on the right disto-lateral corner of the first and second urosomites in females, and on the right caudal ramus on the ventral surface in males.
Diagnosis. Adult female: Cuticle surfaces of prosomal somites smooth dorsal and laterally (Fig. 1A, B). Antennules tip reaching beyond the caudal rami. Right wing of fifth pediger with one tiny dorsal spinule plus one stout ventral spine; left wing with two spines (Fig. 1A, D). No dorsal process on the last thoracic somite (Fig. 1B). Genital double-somite and second urosomite with one lateral wedge each on distal margin on the right side (Fig. 1D). Genital double-somite asymmetric and laterally bulbose; each bulb bearing a stout lateral spine (Fig. 1D). Short spines on the rostrum, which are less than 3 times longer than wide ( Fig. 2A, B). Endopodite of fifth leg 2-segmented with a row of short spinules (arranged in one oblique line) flanked by 2 larger spinules; Exp3 of the fifth leg bearing 2 apical spines ( Fig. 2G).
Adult male: The cuticle surfaces of prosomal somites are smooth dorsally and laterally (Fig. 1C). Right antennule 22-segmented, with one fang-like process on antepenultimate segment, which is less than half the length of the penultimate segment (Fig. 4D). Right antennule with spiniform process on segments 10, 11, and 13 to 16 (Fig. 4D). Inner margin of caudal ramus fringed by long hair-like setae (Fig. 1C). One wedge on distal margin of right caudal ramus on the ventral surface (Fig. 4E). Left and right coxae of the fifth leg have long, acute spines on lateral margins; apical spine of right Exp2 with tiny spinules along medial margin (Fig. 5D). One triangular and one rounded projection located at distal and proximal margins of the right basis, respectively, plus one hyaline membrane on caudal side (Fig. 5D). The aculeus length is almost the width of right Exp2. Left and right endopods one-segmented, with apical spinules (Fig. 5C).
P1-P4: The number of segments on endopods and exopods of P1 to P4, as described for copepods that belong to the Diaptomidae family (Dussart and Defaye 1995). Armature formula of swimming legs as shown in Table 2 including Schmeil's organ on Enp2P2.
Fifth leg (Fig. 2G): Coxa with a large spine on distal margin, basis quadrangular with a blunt projection on distal margin and one slender lateral seta. Exp1 1.66 times longer than 2-segmented endopod. Second endopodal segment with a row of spinules at the tip arranged in an oblique line, flanked by 2 larger spinules. Exp2 separated, with one long and one short spine. Exp3 with spinules on distal medial and lateral margins.
Right wing of fifth pediger with 1 tiny dorsal spinule and 1 ventral spine (Fig. 4B); left wing bearing 1 small spine (Fig. 4C). Urosome: Urosomites nude dorsally and ventrally. First urosomite with thin spine on right side and fold on left side (Fig. 4E). Fourth urosomite slightly projected on right side. Right caudal ramus with wedge-like structure at disto-inner corner of ventral surface; medial margins of rami pilose (Figs 4E, 5A, B). P5: Coxal segments with strong spines on caudal view; left and right basis with a lateral seta (Fig. 5D).
Left basis with a triangular protuberance on distal margin of frontal surface (Fig.  5C). Both left Exp1 and Exp2 pilose on medial margins (Fig. 5F), left Exp2 triangular, with its tip adorned with a small inner seta and spinules (Fig. 5F). Left Enp 1-segmented, distally feathered and as long as left Exp1 (Fig. 5C).
Right basis basally and distally projected: basal projection rounded whereas distal projection triangle-shaped; one semi-triangular sclerotization on caudal surface of right basis (Fig. 5D, E). Right Exp1 quadrangular in frontal view (Fig. 5C), one triangular fold and one rounded projection on caudal view (Fig. 5D). Right Exp2 1.6-1.8 times longer than wide and 1.9-2.0 times longer than aculeus, smooth in both frontal and caudal views (Fig. 5C, D). Aculeus inserted at distal third of the segment, pointed, unarmed, short (no longer than the width of the right Exp2). Terminal claw twice the length of Exp2 smoothly bent and ornamented with tiny spinules on inner margin (Fig. 5D). Right Enp one-segmented slightly longer than right Exp1 (Fig. 5C).
Molecular features. The nucleotide sequence (607 bp) obtained for specimen MAGA-0156 (one adult male), identified as M. cuneatus sp. n. is shown below: GGAGCCTGGTCAGGCATAGTAGGAACAGGCCTTAGAATGAT The K2P maximum distance between the surveyed species reaching 5.52% (Table 3). The nearest neighbour of M. cuneatus sp. n. is M. albuquerquensis with 18.64% of genetic distance (Fig. 6).

Discussion
Mastigodiaptomus cuneatus sp. n. is assigned to the genus Mastigodiaptomus because it fulfils all the morphological generic criteria as given in Dussart and Defaye (1995), especially the left and right antennules of females and males and the ornamentation of coxa, basis and the endopodal or exopodal lengths of the fifth legs. Mastigodiaptomus cuneatus sp. n. (from northern Mexico) appeared to be morphologically close to M. amatitlanensis (from Lago Amatitlán, Guatemala). Similarities between these species in females include the presence of one protrusion on the second urosomite and the bulbose lateral margins of genital double-somite. The similarities in males are the short aculeus and the lack of hyaline membrane on the right Exp2 of the fifth leg.
However, M. cuneatus sp. n. can be separated from M. amatitlanensis by the following features: the dorsal projection on the last pediger absent vs. present; the genital double-somite with vs. without a protrusion on the distal right side; on the genital double-somite, the right spine located at a higher level than the left spine vs. both left and right spines placed at same level; and the endopod of fifth leg long and 2-segmented vs. short and 1-segmented.
The males of these species show more morphological differences in the fifth leg: the caudal surface of the right basis is bulbose with the oblique, medial, angulose and curved cuticular process in M. cuneatus sp. n. in comparison to the rectangular basis with a transversal, distal, cuneiform lamella in M. amatitlanensis. In addition, the distal margins of the left and right endopods bear short setules in M. cuneatus sp. n. whereas in M. amatitlanensis these distal margins bear one slender seta. The aculeus on the right Exp2 is clearly straight in M. cuneatus sp. n. but short, distal and curved in M. amatitlanensis. The Exp2 is smooth in M. cuneatus sp. n. but in M. amatitlanensis an oblique ridge on the caudal surface is present. Finally, we assume that the wedge on the right caudal ramus present in M. cuneatus sp. n. is absent in M. amatitlanensis because there is no mention of a similar feature in Wilson (1941) or Wilson and Yeatman (1959).
Results related with the COI gene suggested that M. cuneatus sp. n. is genetically closest to M. albuquerquensis s. str. and the species recorded in Mexico with one sclerotization (similar to the half wing of a butterfly) on the right basis of fifth leg of males such as M. patzcuarensis. As previously discussed (see Gutiérrez-Aguirre et al. 2014), this particular sclerotization should not be used as a specific character because actually, it is shared by at least three different species. Therefore, in addition to the genetic distance and the sequences of the COI gene, some morphological features are suggested to separate them.
Whereas the distal margins of prosomites are pilose in females and males of M. patzcuarensis, these structures are nude in M. cuneatus sp. n and they have tiny spi- are not yet available for comparison and then the genetic distances between the species showed in Fig. 6 may change when the genetic sequences of the previous species can be added. However, until now, the lowest genetic distance in nearest neighbours of Mastigodiaptomus is between M. patzcuarensis and M. cf. albuquerquensis (3.36%), which probably are cryptic species (see Gutiérrez-Aguirre et al. 2014).
Mastigodiaptomus is considered a Neotropical genus and the species with the wid-

Conclusion
Morphological and genetic differences were found when M. cuneatus sp. n. was compared with the ten known Mastigodiaptomus species, particularly in the female and male urosomes, the male right antennule and fifth legs, and in the COI gene sequence. This report increases the number of recognized species of Mastigodiaptomus to eleven. Mastigodiaptomus cuneatus sp. n. appears to be part of the M. albuquerquensis complex. Second urosomite with a spine-like protuberance on the right disto-lateral corner; one-segmented