Remarks on Mastigodiaptomus (Calanoida: Diaptomidae) from Mexico using integrative taxonomy, with a key of identification and three new species

Background In Mexico, species of four families of free-living calanoid copepods have been recorded as inhabitants of several freshwater systems. These families are Centropagidae, Temoridae, Pseudodiaptomidae and Diaptomidae. The genera Leptodiaptomus and Mastigodiaptomus are the most speciose diaptomid genera in Mexico, and they inhabit natural and artificial lakes, ephemeral ponds, springs, and caverns. Leptodiaptomus is considered as an endemic Nearctic genus, whereas Mastigodiaptomus is a widely distributed Neotropical genus in the southern USA, Mexico, the Caribbean Islands and Central America. Based on new and recent evidence, Mastigodiaptomus diversity has been underestimated: six species of the genus were known before 2000. In this work three new Mastigodiaptomus species have been described from different regions of Mexico by using integrative taxonomy. We also gave amended diagnosis of M. nesus Bowman (1986) and M. patzcuarensis s. str. (Kiefer, 1938). Methods In this work, the taxonomic status of the species was clarified using modern, integrative method based on the COI gene as a DNA marker, plus micro-structural analysis (based on SEM and ligth microscopy). Results Three new species of Mastigodiaptomus were described based on genetic and morphological analyses: M. alexei sp. n., M. ha sp. n. and M. cihuatlan sp. n. Also amended description of M. nesus, morphological variation of M. patzcuarensis s. str., and a comparison of them with all known sequences within the genus are provided. These new findings show that in Mastigodiaptomus differences in several cuticular microstructures of several appendages (such as the antennules, the fifth legs, or the urosomites of these copepods) agree with the interspecific genetic divergence >3% observed in sequences of the COI gene, and the integration of this information is a powerful tool in species delineation.


INTRODUCTION
In Mexico, species of four families of free-living calanoid copepods have been recorded as inhabitants of several freshwater systems. These families are Centropagidae, Temoridae, Pseudodiaptomidae and Diaptomidae.
The genera Leptodiaptomus and Mastigodiaptomus are the most speciose diaptomid genera in Mexico, and they inhabit natural and artificial lakes, ephemeral ponds, springs, and caverns. Leptodiaptomus is considered as an endemic Nearctic genus, whereas Mastigodiaptomus is a widely distributed Neotropical genus in the southern USA, Mexico, the Caribbean Islands and Central America.
Under this assumption, in this paper, three new species of Mastigodiaptomus are described based on genetic and morphological analyses. In addition, amended description of Mastigodiaptomus nesus Bowman, 1986 and M. patzcuarensis (Kiefer, 1938) and a comparison of them with all known sequences within the genus is provided. Finally, a key (based on the morphology) for the identification of the known species of the genus is yielded.

Morphological analysis
Biological samples were obtained by limnetic plankton trawls using a plankton net with 50 mm mesh, and the material was fixed in 96% ethanol. The surveyed samples were collected along a latitudinal gradient from 17 to 24 N and a longitudinal gradient from 87 to 101 W. A full morphological description of adult males and females of the three new species, as well as an amended description of one more species of Mastigodiaptomus, is presented: the morphology of paratype specimens of M. nesus was analysed.
Detailed analysis and illustration of morphology were performed with the aid of a camera lucida, light microscopy and the scanning electron microscopy JEOL-SM-6010, located at El Colegio de la Frontera Sur (ECOSUR, Chetumal Unit) according to Suárez-Morales et al. (1996).
Several analysed specimens were dissected with tungsten needles, and their appendages were mounted in glycerine. The un-dissected biological material was preserved in 100% ethanol with a drop of glycerine. The type material and specimens were deposited in the Reference Collection at El Colegio de la Frontera Sur (ECOCH-CH-Z). then introduced into PGDSpider Vers. 2.1.1.5 (http://www.cmpg.unibe.ch/software/ PGDSpider/) to obtain the nexus file compatible with MrBayes Vers. 3.2.5 and jModelTest 2.1.10. The best fit was HKY+G, and it was used in MrBayes along with 1 M generations to obtain a tree (Supplemental File 2). The general mixed Yule coalescent (GMYC) model was used as a species delimitation method in which the simple threshold approach assumes that there is a threshold time before which all nodes reflect diversification events (interspecific) and after which all nodes reflect coalescent events (intraspecific). The number of species obtained by this approach is thus estimated by this threshold time. The GMYC method was applied in the 'splits' R package for the ultrametric COI tree, obtained with BEAST v1.8.3.

Nomenclatural acts
'The electronic version of this article in Portable Document Format will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank Life Science Identifiers (LSIDs) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix http://zoobank.org/. The LSID for this publication is: urn:lsid:zoobank.org: pub:FEC66E33-E192-41C6-B3E1-C77C5D6705DE. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central and CLOCKSS'.

RESULTS
All the specimens analysed here were classified as species of Mastigodiaptomus and showed the following armature in the trunk limbs: first leg (P1) with 3-segmented Exp and 2-segmented Enp. Trunk limbs P2-P4 with 3-segmented Exps and Enps. Exp1-Exp3 of P1-P4 with one lateral spine (Table 1). In addition, the analysed specimens showed the morphological features that define Mastigodiaptomus according to Kiefer (1932), Wilson & Yeatman (1959) and Dussart & Defaye (1995).
General morphological resemblance of the buccal appendages among species of Mastigodiaptomus was observed in the antenna, mandible, maxillule, maxilla and maxilliped among all the species described here. These resemblances have been described before, supported by the number of segments and the general micro-structure of these Table 1 Ornamentation of prosomal appendages. Armament of swimming legs (P1-P4) in the females and males of the Mastigodiaptomus species described here. Roman numerals indicate spines, and arabic numbers are setae.
Genital double somite slightly longer than wide, bulbous, and asymmetrical: right spine more proximal and larger than the left spine. Parallel rows of tiny spines on anal dorsal plate; hair-like setae along lateral and medial margins of the caudal rami (Fig. 1C).
Coxal segments of male P5 with one long spine each; right coxa bilobed medially (arrow in Fig. 2D); right basipodite rectangular, without proximal-medial protrusion, with one rounded hyaline membrane on the medial margin. Right Exp1 is 2 times longer than the right Enp (Fig. 2D). Straight ridge on right Exp2 laterally projected (Fig. 2E). Distal aculeus 0.7 times the length of the right Exp2.
Left Exp1 of male P5 is 1.7 times longer than the left Enp, and left Exp2 is distally attenuated, almost triangular in shape, hairy (Fig. 2F).
It is noteworthy that females and males from El Padre, Minicenote, and Laguna Cobá (southeastern Mexico) are shorter in body length (0.90-1.0 mm in both sexes) than the paratypes, these latter with a body length from 1.44 to 1.54 mm in females and 1.30 to 1.44 in males (Bowman, 1986).
Paratypes: Two adult females and 20 adult males preserved in 96% ethanol and one drop of glycerine (ECOCH-Z-10318).  Etymology: The species is dedicated to the son of MEG, who has assisted in field collections since the age of 5 years old.
Additional material: Two adult females and males for genetic analysis from wetland near Camalote, Veracruz, Mexico. Three adult females and three adult males for SEM analysis from the same place.
Diagnosis: Female prosomites and urosomites dorsal and laterally smooth, genital somite slightly asymmetric, lateral borders almost parallel, antennules reaching the furcal rami, and furcal rami with long setules along both lateral and medial margins (Fig. 3A). Lateral wings of the last prosomite projected, with two spines on each side; left wing more distally projected than the right wing (Figs. 3C and 3D). Antenna with 2-segmented Enp, Enp2 with two lobes: medial lobe with 9 setae, terminal lobe with 7 setae. Praecoxal arthrite of maxillule with 9 acute setae on frontal group (+4 setae on caudal group). Two praecoxal lobes on maxilla; first lobe with 4 setae, second lobe with 3 setae. Three-segmented maxillar Enp, with 1, 1 and 3 setae respectively. Similar to that in female, the males bear two spines on each lateral wing of the last prosomite (Figs. 3E and 3F), and the antenna, maxillule and maxilla as previously described for females. First male urosomite with a long spine on the right side, directed distally ( Fig. 3F). Right Exp2 of fifth male leg with one short distal aculeus, one sclerotisation on the caudal surface, and one long distal spine. This distal spine, slightly longer than the length of the right leg, is curved and directed towards the distal region ( The second and third prosomites are the widest regions of the body; the left wing of the last segment is more projected towards the distal region of the body than the right wing; antennule, reaching the distal margin of the caudal rami ( Fig. 3A).
Right and left wings of the last prosomite with dorsal and ventral spines directed laterally (Figs. 3C and 3D). Genital somite asymmetric, 1.6-1.8 times longer than wide; left margin almost straight, with a strong spine placed more distally than the right spine, the latter spine inserted on the slightly protruding right margin (Figs. 3A and 5C). Distal, ventral surface of the genital double somite with deep scars on the cuticle, which are arranged in a parallel pattern (arrow, Fig. 5C). Anal somite with lateral folds (arrow, Fig. 5D) and hair-like setae along the lateral and medial margins of the caudal rami ( Fig. 5D).
Fifth leg: Coxa with left and right lateral spines (Fig. 5E); 2-segmented Enp, which bears 2 long setae and a row of short setules apically (Fig. 5F). The Enp is 0.8-0.9 times the length of the inner margin of Exp1. Second exopodite with spines along the margins and one lateral, short spine placed next to Exp3. The latter is separated and bears one long plus one short spine (Fig. 6A).
Male: 1.1-1.3 mm body length. As in females, the cuticular surfaces of the pro-and urosomites are smooth (Fig. 6B). Left male antennule as in female; right geniculate antennule 22-segmented, with a spiniform process on segments 10, 11 and 13-16. Spiniform process on segment 10 reaching the distal margin of the bearing segment. Spiniform process on segment 11 reaching the distal margin of segment 12 (Figs. 4A and 6C).
Right fifth leg, caudal view: Coxopodite with a large lateral spine, this segment strongly sclerotised distally and bent to the left. Basipodite folded distally, with a lateral short seta and one rounded hyaline membrane on the medial margin; one protrusion present on the mid-caudal face of the basipodite, which is very high (Figs. 4F, 6D and 6E). Exp1 with a ridge on the medial margin (arrowed in Figs. 6E and 6F) and with one angled lamella laterally. Endopodite is twice the length of the medial margin of Exp1.
Exp2 is 1.7-1.8 times longer than wide, with crescent-shaped sclerotisation; this exopodal segment is 1.1-1.2 times longer than the aculeus. This aculeus is placed distally and directed posteriorly, as is the strongly curved distal spine (Fig. 3E). The distal spine is 1.1-1.2 times longer than the entire right leg.
Distribution: Mexico, found only in a wetland near Camalote, Veracruz (the type locality).  Holotype: One adult female dissected on a semi-permanent slide (ECOCH-Z-10319).
Paratypes: 20 adult females and 20 males preserved in 96% ethanol and one drop of glycerine (ECOCH-Z-10321). Etymology: The species name is a noun in apposition, and the term 'ha' means 'water' in the Mayan language.
Additional material: Five adult females and five adult males for genetic analysis from Verde Lucero (20 52′09.57″ N; 87 04′37.52″ W) and 7 Bocas. Five adult females and five  Diagnosis: Female with short antennules, reaching the second urosomite; fifth prosomite with or without a dorsal projection; and the left wing of the fifth prosomite slightly projected distally (Fig. 7A). Antenna with 2-segmented Enp, Enp2 with two lobes: medial lobe with 9 setae, terminal lobe with 7 setae. Praecoxal arthrite of maxillule with 11 acute setae on frontal group (+4 setae on caudal group). Two praecoxal lobes on maxilla; first lobe with 4 setae, second lobe with 3 setae. Three-segmented maxillar Enp, with 1, 2 and 3 setae respectively. Genital double somite clearly longer than wide, almost symmetric, but the right lateral spine is inserted more proximally than the left spine (Fig. 7F). Wrinkles on the ventral surface of the genital double somite and anal somite (Figs. 7F and 7G). Caudal rami with hairy lateral and medial margins (Fig. 7G). Endopod of fifth leg two-segmented and slightly shorter than the Exp1 of the fifth leg (Fig. 7H). The right and left prosomal wings of males with spines; first urosomite with a lateral, strong spine on the right margin (Fig. 7B); right male antennule with a weak, short spine on segment 8 but a long spinal process on segments 10, 11 and 13-16 (Fig. 8A). Antenna, maxillule and maxilla as described for females. Pre-anal segment with a rounded, right protrusion and caudal rami with the medial margin hairy in males (Figs. 8B and 8D). Right fifth male leg with a basipodite bearing one basal protrusion and one basal hyaline membrane, right Exp2 almost rectangular, with frontal and caudal surfaces smooth and bearing one straight distal aculeus (Figs. 8F and 8G). Long left Exp1, is 2.6 times longer than the left Exp2 (Fig. 8F).
Female: 1.2-1.3 mm body length, including the caudal rami. Holotype without a dorsal process on the last prosomite (Fig. 7A), but in 20% of female paratypes, this process is present (Fig. 9C). The second prosomite is the widest region of the body. In comparison with the right wing, the left wing of the last segment is slightly more projected towards the distal region of the body; antennule reaches the half length of the anal somite (Fig. 7A). Rostrum with pointed ( Fig. 7C) or rounded spines (Fig. 9A). Latero-ventral and dorsal surfaces of prosomites smooth, without ornamentation (Fig. 9B). Antennules 25-segmented, each segment armed as follows: (1)  Right and left wings of the last prosomite with dorsal and ventral spines directed laterally (Figs. 7D and 7E). Lateral margins of the genital double somite almost parallel, 1.6-1.7 times longer than wide; left margin almost straight, with a strong spine placed slightly more distally than the right spine ( Fig. 7A and 7F). Genital field quadrangular (Fig. 9D). Distal, ventral surface of the genital double somite with deep scars on the cuticle curved, parallel, sometimes forming a pattern similar to fingerprints (Figs. 7F and 9E). Anal somite with lateral folds (arrow Fig. 7G) and hair-like setae along the lateral and medial margins of the caudal rami (Fig. 7G).
Fifth leg: Coxa with lateral spines (Fig. 9F); 2-segmented Enp, which bears 2 long setae and a row of short setules apically (Fig. 7H). The Enp is 0.8-0.9 times the length of the inner margin of Exp1 (Fig. 7H). Second exopodite with spines along the margins and one short lateral spine placed next to Exp3. The latter is separated and bears one long plus one short spine (Fig. 10A).
Male: 1.2-1.3 mm body length. Cuticular surfaces of pro-and urosomites smooth (Fig. 7B). Rostral spines slightly more pointed than in the female (Fig. 10B). Right geniculate antennule 22-segmented, with spiniform process on segments 10, 11 and 13-16 (Fig. 8A). Spiniform process on segment 10 reaching the distal margin of the bearing segment. Spiniform process on segment 11 reaching the distal margin of segment 12 and 1.5 times longer than the width of the bearing segment (Fig. 8A).
Spiniform processes of antennular segments 15 and 16 projecting from the mid-length of the bearing segment and spiniform process of antennular segment 16 similar to a flat, hollow cavity (Fig. 10C). Antepenultimate antennular segment with a hook-like projection that reaches the distal third of the penultimate antennular segment (Fig. 10D) and with a lateral hyaline membrane (Fig. 10E, arrow).
Left wing of the last prosomite with one small spine and one small setule in the ventral and dorsal positions, respectively (Fig. 10F). The spine and seta on the right wing of the last prosomites are larger than those on the left wing.
First urosomite with one spine on the right margin (Figs. 8B and 8C); pre-anal somite with a rounded protrusion on the distal, dorsal right corner, caudal rami pilose medially (Fig. 8D); right ramus with a bulbous hump on the ventral surface (Figs. 8D and 8E).
Right fifth leg, caudal view: Coxopodite with a large lateral spine. Basipodite folded distally, with a lateral short seta and one rounded hyaline membrane on the medial margin; one protrusion present on the mid-caudal face of the basipodite (Fig. 8F), which is low (Fig. 8G, arrow Fig. 10G). Exp1 with rounded lamellae on the medial and lateral margins ( Fig. 8F; arrow, Fig. 10H). Endopodite is twice the length of the medial margin of Exp1. Exp2 is 1.6-1.7 times longer than wide, with a smooth surface (Figs. 8F and 10H); this exopodal segment is 1.4-1.5 times longer than the aculeus. This aculeus is placed distally (Fig. 8F), as is the strongly curved distal spine; the distal spine is as long as the entire right leg (Fig. 8H).
Paratypes: One adult male dissected on three semi-permanent slides: prosome and urosome (slide 1); A1, A2, P1-P4 (slide 2); P5 (slide 3) (ECOCH-Z-10324). Fifteen adult females and 11 adult males preserved in 90% ethanol and one drop of glycerine (ECOCH-Z-10325). Etymology: The species name is a noun in apposition, and the term 'cihuatlan' means 'women's place' in the Nahuatl language, which is an original language in Guerrero state. The name is dedicated to the sisters of MAGA.
Additional material: Two adult females and five adult males for genetic analysis from the type locality.
Diagnosis: Widest region of the female body on the second and third prosomites; long antennules: 6-7 antennular segments beyond the caudal rami. Antenna with 2-segmented Enp, Enp2 with two lobes: medial lobe with 7 setae, terminal lobe with 7 setae. Praecoxal arthrite of maxillule with 10 acute setae on frontal group (+4 setae on caudal group). Two praecoxal lobes on maxilla; first lobe with 5 setae, second lobe with 3 setae. Three-segmented maxillar Enp, with 1, 2 and 3 setae respectively. Genital somite asymmetric, right margin strongly acute and left margin rounded. Second urosomite partially segmented: segmentation between the second and anal urosomites, only evident dorsally (Fig. 11A). Female fifth leg strongly armed with a long spine on both coxae; this spine is almost as long as the medial margin of the basipodite (Fig. 11C). Fifth-leg coxae with a deep fold extending from the medial margin to the frontal surface (Figs. 12H and 12I).
Widest region of the male body at the level of the second and third prosomites. Antenna, maxillule, and maxilla as described for females. Asymmetric urosomite: the anal somite and both left and right rami twisted to the right (Figs. 11B and 11D). Medial margin of caudal rami hairy, but the right caudal ramus with gaps between the hairs (Figs. 11D and 11E). Dorsal surfaces from second to fourth urosomite with tiny spines (Fig. 11D). Right male antennule 22-segmented, with a strongly developed spiniform process on segments 8, 10, 11 and 13-16; antepenultimate antennular segment with one angled projection as long as the middle length of the penultimate segment (Fig. 11B). Male right fifth leg with one angulate projection on a basal bulbous widening on the right basipodite, plus a long hyaline membrane along the medial margin of the same segment. Right Exp2 with two rounded projections, one aculeus slightly longer than the bearing segment, and one long, acute apical spine (Fig. 11F).
Genital somite asymmetric: right margin acute, bearing one large, apical spine; left margin rounded, with a short spine placed more dorsally. Genital somite slightly twisted towards the postero-dorsal region. Hair-like setae along the lateral and medial margins of the caudal rami, the lateral setae slightly thicker than the medial setae (Fig. 12E). Urosome 2-segmented ventrally (Fig. 12F): separation between the second and anal somites only evident lateral and dorsally (Fig. 12G).
Fifth leg: Quadrangular coxa, one large coxal spine as long as the middle length of the lateral margin of the bearing segment (Figs. 11C, 12H and 12I). From the base of the coxal spine to the frontal coxal surface, a long fold runs along the lateral coxal margin (arrow,Figs. 12H and 12I). Fifth leg with 2-segmented Enp, which bears 2 apical, long setae and short setulae diagonally arranged. On average, EnpP5 is 1.1-1.2 times longer than the medial margin of Exp1P5 (Fig. 12H). Exp2 with a row of strong spinules along both margins; one lateral, short spine placed next to Exp3. The latter is separated and bears one long plus one short spine (Fig. 11C).
Male: 0.97-1.2 mm body length, including the caudal rami. Rostral spines long, acute (Fig. 13C). Cuticular surface of prosomites smooth; tiny spinules on dorsal surfaces of urosomites (Fig. 11D). First urosomite with a large spine on the right lateral margin (Fig. 13D) and one tiny hair-like setule on the left margin (Fig. 13E). Anal somite and caudal rami twisted to the right side; right caudal ramus with angular polygonal thickenings on dorsal and ventral surfaces, arranged as keels; medial margin of right ramus less pilose than medial margin of left caudal ramus (Figs. 11D and 11E).
Right geniculate antennule 22-segmented, spine on segment 8 modified into a long spiniform process that reaches a third of the length of segment 9; also a spiniform process on segments 10, 11 and 13-16 (Fig. 13A). Spiniform process on segment 10 reaching the half length of the next segment. Spiniform process on segment 11 reaching the distal margin of segment 12 (Fig. 13A).
Right wing of the last prosomite with one thin dorsal seta and one large ventral spine (Fig. 13D); left wing marginally smooth (Fig. 13E).
Right fifth leg, caudal view: Coxa with one lateral spine (Fig. 11F). Basipodite bulbous and with one angled basal process (process arrowed in Figs. 13F-13H), plus a long hyaline membrane that extends along the medial margin of the segment (Figs. 11F, 13F and 13G). Right Exp1 with a triangular, lateral apex and two rounded lamellae on the medial margin (Fig. 11F). Right Enp1 slightly longer than the medial margin of the right Exp1 (Figs. 13F and 13G). Lateral margin of Exp2 curved; surface of the right Exp2 with two sclerotised (basal) projections (Figs. 11F, 13F, 13H and 13I) and one sclerotised line that forms a small triangular hump distally (Figs. 13F and 13H). One straight aculeus inserted in half of Exp2 and longer than the bearing segment, plus one long acute apical spine, which is as long as the entire right P5 (Fig. 13H).
Last prosomite projected on left side, with two large spines. Genital double somite is 1.3-1.4 times longer than wide, clearly asymmetrical: right margin laterally acute and with a strong spine inserted more proximally than the left spine, which is on a sinuous margin (Fig. 14E). Left spine of genital double somite inserted at 45% of the segment length. Some parallel, curved cuticular sclerotizations near posterior margin of the genital double somite and pre-anal somite (arrow, Fig. 14E). Caudal rami with hair-like setae along the medial margin and spine-like setules along the lateral margins (Fig. 14F).
Left wing of last prosomite with one large spine ventrally and one thin seta dorsally; first urosomite with one short setule placed laterally on the left margin. Right wing with two spines, the ventral spine larger than the dorsal spine; first urosomite with a large spine placed distally on the right margin (Figs. 15E and 15F). Ventral and dorsal surfaces of all urosomites smooth; distal margin of the fourth urosomite slightly projected on right side. Caudal rami pilose medially; right ramus with a bulbous hump on the ventral surface (Fig. 15F).
Right fifth leg, caudal: The intercoxal sclerite, and the coxa folded towards the left but not strongly projected. Coxa with one lateral spine, one tiny bi-lobed fold, and one tiny rounded protrusion (arrowed in Fig. 15G). Basipodite basally protruded, with lateral seta and with two hyaline membranes in caudal view: one rounded, medial; and one angled, distal. Enp slightly longer than Exp1; the latter with rounded protuberances on the medial and lateral margins. Right Exp2 with one curved, medial hyaline membrane and one basal angulate thickening. In the distal third, a curved aculeus is inserted, which is shorter than the bearing segment: ratio between the length of Exp2 and the length of the aculeus = 1.2 (Fig. 15G). Curved distal spine is 2.4-2.5 times longer than Exp2.
Left fifth leg: Coxa with one large, lateral spine (Fig. 15G). Basipodite with one lateral seta, one tiny, rounded projection in the basal region, plus an inverted Y-shaped wrinkle (arrowed in Figs. 15G and 16D), which produces a cuticular thickening in the next segment, visible with light microscopy. Endopod and Exp1 with similar lengths, the latter medially pilose. Exp2 triangular, distally angulate, medially pilose, with a delicate distal seta and 2 or 3 tiny spinules (Fig. 15G

Genetic analysis
DNA analyses revealed 16 clades within the genus (Fig. 17) based on a total of 203 sequences that were collapsed into 86 different haplotypes by ALTER software. All records were downloaded from the BOLD database and verified for correct identification. After alignment, we obtained a maximum length of 571 bp, and one sequence was 468 bp in length. With the exception of this sequence, all the sequences were over 500 bp in length. The average distance within species of the genus was 2.31% and the maximum 12.19%, this latter was observed in M. montezumae (Brehm, 1955), which requires further study for clarification. Mastigodiaptomus siankaanensis Mercado-Salas, Khodani, Kihara, Elías-Gutiérrez and Martínez-Arbizu, 2018, was splitted in two clades and one entity, due to the addition of more sequences than a previous study (Mercado-Salas et al., 2018). They also require a more detailed analyses of south and north populations in Yucatan Peninsula.
Divergences within species of the five taxa analysed here are included in Table 2. In all species, values were under the 3.0% threshold, except in M. patzcuarensis, the only species with a wide distribution in the Mexican Plateau with two BIN assignments. In the case of M. patzcuarensis, the maximum was 4.78%, and it was possible to distinguish two sub-clades, but there was no biogeographical justification for separate them because the populations seemed to be intermixed, possibly due to passive dispersal of resistant eggs. The GC content ranged from 38.76 ± 0.2% to 45.51 ± 0.3% in the five studied species (Table 2), and all values were near the range for all species of the genus (42.77 ± 0.14).
The number of species suggested by the GMYC analyses with a confidence interval of 11-19 gave 12 species plus 4 entities (species inferred from one sequence) consistent with the clades from the Bayesian analyses. The likelihood of GMYC model (539.65) was significantly superior to the null model (535.23) (see Fig. 17).

DISCUSSION
The first diaptomid species described in Mexico as a new species belongs to the genus Mastigodiaptomus; it was called M. lehmeri Pearse, 1904. Later, this species was synonymised with M. albuquerquensis by Marsh (1907). Suárez-Morales & Elías-Gutiérrez (2003) described this process in detail.
In Mexico, Mastigodiaptomus has been recorded in water systems from between 1,600 and 2,400 m.a.s.l. in the Neovolcanic Axis Province (Dos Santos-Silva, Elías-Gutiérrez & Silva-Briano, 1996) to other regions, such as semi-desertic provinces, including the Mexican Altiplano (Gutiérrez-Aguirre, Cervantes-Martínez & Elías-Gutiérrez, 2014). The genus is also found in surface waters in the Caribbean Province at lowlands (Bowman, 1986;Mercado-Salas et al., 2018). This Neotropical genus is clearly able to inhabit a wide range of habitats. Therefore, Mastigodiaptomus is the diaptomid genus with the highest diversity in Mexico: the number of currently known species (including the ones described in this paper) is higher than the number of Leptodiaptomus species (13 vs. 7, respectively) and is comparable to the number of species of two other freshwater, free-living cyclopoid genera also distributed in  (Kiefer, 1932;Wilson & Yeatman, 1959;Dussart & Defaye, 1995) are: segment 11 of the antennules of females and left antennule of males with two setae; coxa of female P5 with a long, spatulated seta; one lateral spine on Exp1P1 in females and males; Enp of right male P5 longer than the right Exp1; right male antennule with a spiniform process on segments 10, 11 and 13-16; and basis of the right male P5 with hyaline lamellae on the caudal side.
Morphological features such as the presence of one lateral spine on Exp1 of P2-P4, the fused Exp2-Exp4 antennal segments, the presence of one seta on antennal Exp1, the Exp2P5 as one unarticulated spine-like process in females, or the right geniculate A1 and P5 in males have supported the monophyletic clade of Diaptomidae and related families in earlier phylogenetic studies by Park (1986) and Bradford-Grieve et al. (2010).
Recently, a multi-gene phylogeny, that includes analysis of COI, is consistent with that monophyletic relationships at Family and Superfamily level (Blanco-Bercial, Bradford-Grieve & Bucklin, 2011) and according with the performed analysis, all these features are present in the species of Mastigodiaptomus.
Mastigodiaptomus alexei sp. n. and M. ha sp. n., are similar to M. texensis described by Wilson (1953), but several features can be used to differentiate the species. The following features can be useful to differentiate females: in M. texensis, the antennule is longer than that in M. alexei sp. n. and M. ha sp. n; when the length/width ratios of the genital double somite are compared, that of M. texensis is the shortest, followed by that of M. ha sp. n., and that of M. alexei sp. n., is the longest. The symmetries of the genital somite are different: in M. texensis, it is symmetric, and the positions of the left and right spines are at the same level in the segment, while in M. ha sp. n. and M. alexei sp. n., the right spine is positioned more proximally than the left spine.
In males, features of the right antennules and fifth legs can differentiate species: the length of the spinal processes on segment 10 and 11 of the right A1 is shorter in M. texensis than in M. alexei sp. n. and M. ha sp. n. The position of the spinal process on segment 16 is more distal in M. texensis than in M. alexei sp. n. and M. ha sp. n. The length ratio between left Exp1P5 and left Exp2P5 is highest in M. texensis, intermediate in M. ha sp. n., and lowest in M. alexei sp. n. The caudal surface of the right Exp2P5 is smooth in M. ha sp. n. but with one crescent-shaped sclerotisation in M. texensis and M. alexei sp. n. The ornamentation of the male right basis of P5 is different among species (see Supplemental File 3). Therefore, a combination of at least 20 morphological features was observed to differentiate among the species analysed.
Some morphological features of M. cihuatlan sp. n. are unique in the genus and can be useful in classifying the species: in females, the asymmetric genital double somite is twisted; the ventral spine is dorsally displaced on the fifth right wing; the anal and pre-anal somites are fused ventrally, and a large fold extends from the caudal, medial margin to the frontal surface of the fifth leg coxopodite. In males, unique features that are useful for distinguishing M. cihuatlan sp. n. from other Mastigodiaptomus species include a spiny, asymmetric, twisted urosomite; the spine of the eight antennular segment modified into a strong spiniform process; and the complex ornamentation of the right basis of the fifth leg.
Detailed morphological analysis performed here revealed that M. nesus is a species with extremely small ornaments on the prosoma and urosoma of adult females and males (see Figs. 1 and 2). This feature was not noted in the original description of M. nesus by Bowman (1986) but was detected in the populations of San Salvador Island (type locality) and was a stable character in all the populations from Quintana Roo surveyed here.
Cuticular surface ornamentation of the body is also observed in the females of M. albuquerquensis (Herrick, 1895), females and males of M. patzcuarensis (Gutiérrez-Aguirre, Cervantes-Martínez & Elías-Gutiérrez, 2014) and males of M. suarezmoralesi (Gutiérrez-Aguirre & Cervantes-Martínez, 2013) whereas M. siankaanensis bears hair-like setulae only on one cephalic appendage, the female antennulae (Mercado-Salas et al., 2018). In the Americas, only two species belonging to Leptodiaptomus have been recorded as having cuticular ornamentation on the dorsal surfaces of urosomites. They are L. siciloides (Lilljeborg, 1889) and L. connexus (Light, 1938), which are distributed from the western and southwestern USA to the northern region of Mexico. We consider that this kind of ornamentation should be considered in further descriptions.
The number of elements on each segment of A1 is stable specially in females when different species of Mastigodiaptomus are compared. However one modified seta replaces one seta, in the antennular segments 18, 21 and 24 in M. alexei sp. n. or in the segment 24 in M. cihuatlan sp. n.
In males more differences were observed (in the species surveyed here) when the number of elements of each antennular segment are compared. For instance, the segment 9 bears two elements in M. alexei sp. n., whereas in the other species, three elements were observed. Mastigodiaptomus nesus lacks one aesthetasc in segment 13; or in M. nesus and M. patzcuarensis the segment 18 bears one modified seta; but in M. ha sp. n. there is one spine, or in M. alexei sp. n. and M. cihuatlan sp. n., the segment 18 has not elements. Then, this kind of ornamentation also seems informative to define morphologically to Mastigodiaptomus species.
The results for these new species confirm the results of previous analyses of the genus (Mercado-Salas et al., 2018), who analysed 11 species, with one exception. The GMYC analysis suggests that the two haplotypes of M. siankaanensis found in her work could represent two different species, plus a third one represented by a singleton. Also, further analyses of M. cf. montezumae and M. cf. patzcuarensis are needed (mainly of populations from semi-desert pools).
Although the three new species are represented by few sequences (1)(2)(3)(4)(5), they are well supported, not only by the DNA barcodes. The conclusion based on morphological diagnostic characters is consistent with that based on GMYC analyses and the BIN system of the BOLD database (see Supplemental File 2) for these three species (Ratnasingham & Hebert, 2013).
According to the K2P analysis, the minimum and maximum interspecific distances between COI sequences within Mastigodiaptomus are 5.21 and 26.48%, respectively, which are consistent with the findings of Mercado-Salas et al. (2018). As shown in Fig. 17, M. patzcuarensis is a well-defined clade, but among all the sequenced species of Mastigodiaptomus, this is the species with the highest intraspecific genetic divergence; the population (females and males) from Chapultepec Lake exhibited almost all of the morphological features previously described as typical for M. patzcuarensis (Gutiérrez-Aguirre, Cervantes-Martínez & Elías-Gutiérrez, 2014); however, one morphological feature was notably variable between populations. The presence of rows of hair-like setae on the prosomites is continuous in the Pátzcuaro, Flor del Bosque, and Ignacio Ramírez populations, but interrupted in the Chapultepec population and lacking in La Goleta, La Cruz and Cuitzeo populations. Additionally, the population from Chapultepec has extremely small rounded projections on the right coxa and left basis of the male fifth leg (arrows in Fig. 15G), which were not observed in the rest of the mentioned populations of M. patzcuarensis. Further studies are necessary to establish whether these genetic and morphological variations reflect reproductive isolation.
Two others rare Mastigodiaptomus recently described are M. cuneatus Gutiérrez-Aguirre & Cervantes-Martínez, 2016, found only in a lagoon within the city of Mazatlán (Mexico) and M. suarezmoralesi Gutiérrez-Aguirre & Cervantes-Martínez, 2013, described from the unique Montebello lakes in Chiapas state, Mexico. The latter species still has not been studied with DNA barcodes. Other species in need of attention with a restricted distribution are M. purpureus Marsh, 1907, found in Cuba, and M. amatitlanensis M. S. Wilson, 1941, from the Amatitlan Lake (Guatemala) (Wilson & Yeatman, 1959). The latter species may be almost extinct because it has not been detected in several surveys of this lake (A. Cervantes-Martínez, 2019, personal communication).
The next goal is understanding the distribution of these new species because after extensive samplings in Mexican freshwater systems, they appear to have only a very restricted distribution. This case is not rare because M. maya Suárez-Morales & Elías-Gutiérrez, 2000; which may be the largest representative of the genus, with a length of more than 2.3 mm (excluding the furcal rami) (Suárez-Morales & Elías-Gutiérrez, 2000), has not been observed since its original description, even in the type locality, an ephemeral pool in a forest (M. Elías-Gutiérrez, 2019, personal communication).
Finally, the level of analysis presented here, considering the specimen number and species number analysed with the COI marker of the Mastigodiaptomus genus is only comparable to that of the studies on Moina (Cladocera: Anomopoda), another microscopic freshwater crustacean distributed in Mexico and the World (Elías-Gutiérrez et al., 2019; Montoliu-Elena, Elías-Gutiérrez & Silva-Briano, 2019). In these cladocerans, the intraspecific distances ranged from 3.72 to 18.09% and thus were lower than the intraspecific distances found for Mastigodiaptomus.

CONCLUSIONS
These findings show that in Mastigodiaptomus, not only the morphological differences in right male antennule, or the fifth legs but also several morphological cuticular characters agree with the interspecific genetic differences higher than 3% observed in sequences of the COI gene.