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Tardigrades from Iztaccíhuatl Volcano (Trans ‐ Mexican Volcanic Belt), with the Description of Minibiotus citlalium sp. nov. (Eutardigrada: Macrobiotidae)

: The study of tardigrade diversity in Mexico is at early stage of development, to date, 56 extant species have been reported. To identify the tardigrade fauna associated with mosses in the Iztaccíhuatl volcano, we performed a systematic sampling along an altitudinal and multi ‐ habitat gradient. A total of 57 moss samples were collected, 233 adults, 20 exuviae, and 40 free ‐ laid tardigrade eggs were extracted from them. Five species were identified, and three putative species were determined. Diphascon mitrense and Minibiotus sidereus represents new records for Mexico and North America, while Adropion scoticum is a new record for Mexico. Additionally, one new species, Minibiotus citlalium sp. nov. was discovered; it resembles to Min. constellatus , Min . sidereus and Min. pentannulatus by the presence of a similar distribution pattern of star ‐ shaped pores in the dorsal cuticle arranged in 11 transverse rows, which become double in the segments of the legs I–III, and by a very large star ‐ shaped pore (5–6 tips) on each leg of the fourth pair. Minibiotus citlalium sp. nov. differs from other Minibiotus species mainly by macroplacoid length sequence, presence of both small and large star ‐ shaped pores on the external surface on all legs, and by egg processes with inconspicuous ornamentation.


Introduction
Tardigrades are ubiquitous micrometazoans present in every biome on Earth, as they can be found in marine, freshwater, and terrestrial habitats [1]. Ecological studies dedicated to tardigrades are scarce, especially those that depend on replicated quantitative samples [2] (pp. 163-210). A patch distribution has been suggested even in apparently identical microhabitats. Therefore, a high number of samples (from hundreds to more than 1000) are necessary to reduce the standard error of population estimates and to obtain valid data for testing ecological hypotheses [2][3][4][5]. Tardigrade taxonomy is currently the subject of investigations, most of them relying on increasingly complex molecular datasets and extensive taxonomic contexts [6][7][8]; nonetheless, the taxonomic studies on Mexican tardigrades are at early stage. These include exploration, intensive collections, detailed observations, description, and denomination of species [9]. Previous studies on tardigrades in Mexico

Study Site
Fifty-seven moss samples were collected in the southwestern slope of Iztaccíhuatl volcano (Sierra Nevada, Trans Mexican Volcanic Belt; Figure 1a) in January 2018. The mosses were sampled in 12 geographical stations (S1-S12) distributed along an altitudinal gradient (2700-4500 m asl; Figure  1b) and in at least five different vegetation types, including: Pinus-Cupressus-Quercus mixed forest; Abies religiosa, and Cupressus sp. forest; Pinus hartwegii forest; alpine scrub; and alpine tundra ( Figure  1c, Table 1). In each station, different moss morphotypes were identified and a square of 2 × 2 cm was sampled from each moss cushion and characterized depending on the collecting site, vegetation, and substrate type (rock, tree bark, and soil).

Sample Processing
Each moss sample was stored inside a paper envelope and dried. In the laboratory, the samples were rehydrated with 20 mL of tap water for 48 h. Later, samples were shaken and rinsed, and the supernatant were filtered using two stacked sieves with a decreasing mesh diameter (100 μm and 74 μm). The retained contents on the 74 μm sieve were washed into a Petri dish for examination under a stereoscopic microscope using dark field illumination at 45 × magnification.
All specimens for light microscopy were mounted individually onto microscope slides in Heinze polyvinyl alcohol (PVA) medium. Observations and photographs were taken using phase contrast microscopy (PCM) (ZEISS Axioskop with digital camera Axiocam ERC 55); for each species, images were recorded at successive focal depths and automatically combined into a single sharp image (i.e., focus stacking).

Morphometrics and Morphological Nomenclature
Structures were measured provided their orientations were suitable; body length was measured from the anterior to the posterior end of the body, excluding the hind legs. Terminology for the structures within the bucco-pharyngeal apparatus follows that from Pilato [33][34][35], Dastych [36], Kaczmarek and Michalczyk [37]. Macroplacoid length sequence is presented according to Kaczmarek et. al. [38]. Claws of Hypsibioidea were measured with the protocols as indicated in Beasley et. al. [21], whereas claws of Macrobiotoidea were measured with the protocols as indicated in Kaczmarek and Michalczyk [37]. The pt ratio (which is the ratio of the length of a given structure to the length of the buccal tube), is expressed as a percentage [33] and its values are always provided in italics. This was done to distinguish them from absolute measurements in micrometers. Morphometric data were compiled using the Parachela Excel template version 1.6, which is available from the Tardigrada Register (http://www.tardigrada.net/register/submit.htm) [39].

Description of Minibiotus citlalium sp. nov.
The identity of specimens of genus Minibiotus was based on the description of Schuster et. al. [55] amended by Claxton [40]; both included in the definition of this genus by Pilato and Binda [56]. The characters observed in our specimens and diagnostic of this genus were presence of buccopharyngeal apparatus of the Macrobiotus type, Minibiotus variant: an antero-ventral mouth, a rigid, short and narrow buccal tube, short ventral lamina (pt < 62), stylet supports inserted at 73% or less of the buccal tube length, pharyngeal apophyses and placoids present, stylet furcae typically shaped, short macroplacoid row length (pt < 42), and claws of the hufelandi type with lunules present. The identity of the Minibiotus citlalium sp. nov. was based on a character matrix of all described species of genus Minibiotus, which was built from the information contained in the original published descriptions. The characters used for the matrix were eyespot pigmentation, cuticular sculpturing, presence/absence of pores, pore shape, arrangement of pores, and pore size. Furthermore, the specimens were compared with the original descriptions of the most similar species: Minibiotus vinciguerrae Binda and Pilato, 1992 To obtain more specimens and eggs of Minibiotus citlalium sp. nov. new moss samples were collected from the same stations and substrates (Table 1) where Minibiotus spp. were found (XX, Table  1). The samples were processed following the method described by Stec et. al. [57]. Ornamented eggs were individually placed in Petri dishes with mineral water at room temperature while waiting upon hatching.
Hatched specimens and eggs were used for light microscopy and processed as previously described; the eggs were measured based on Kaczmarek and Michalczyk [37]. For further refinement of initial observations, a subset of these material from the new species were processed for scanning electron microscopy (SEM). Briefly, specimens were first boiled in absolute ethanol and transferred to cold absolute ethanol. This was repeated three times. Then they were boiled again in absolute ethanol until complete evaporation. Finally, the specimens were mounted on metal plates and covered with gold. Specimens were examined in a Hitachi Scanning Electron Microscope S-2469N.
The description of Minibiotus citlalium sp. nov. was based on 16 animals and three eggs, one of them still hatching. In the description of Min. citlalium sp. nov. the diagnosis of the species and the differential diagnosis are included; both contain ranges of variation for some characters, while for others, the specific information obtained from the descriptions or from the images of the holotypes shown in the respective descriptions is included.
Information about diagnostic characters was consulted with the authors of the most similar species [58] Table 2. Because pt index only satisfactorily eliminates body size effects for isometric traits but do not eliminates from allometric ones [59,60]; we evaluate growth relation (isometric vs. allometric) in the continuous traits respect to body size in Minibiotus citlalium sp. nov., following the methodology proposed by Bartels et. al. [60]. The effect of "body size", measured as buccal tube length (BTL), respect to 26 of 29 continuous body traits was evaluated, by mean a linear regression in each (Supplementary Material Table S1). Regressions were performed from log transformed values, omitting certain individuals if their orientation was unsuitable. The isometric or allometric trend was determinate in each trait, comparing the slope of each linear regression with a slope of 1; to do this, we performed t-tests (t = (b)1)/SE of the slope, df = n)20) [61]. Regression analysis were carried out using PAST ver. 4.03 [62]. We provide supporting data for growth trends, as well the slope (b) and the Y intercept (a) for each trait analyzed. It has been suggested that pt index for each body trait should be reported together with the slope (b) and the Y intercept (a), both obtained from the regression of logBS (body size) vs. logY (Y = body trait). Whit these parameters Thorpe's normalization can be performed yield any quantitative trait size-independent [60].

Results
Tardigrades were observed in 32 out of the 57 samples examined (ca. 56%). In total, 233 tardigrades, 20 exuviae, and 40 free-laid eggs were found. Only 13 eggs were identified at a specific level. The tardigrades belong to seven genera within three eutardigrade families: Calohypsibiidae:

Remarks: The specimens correspond to the key proposed for the genus by Michalczyk and
Kaczmarek [65] and the most recent revision of Calohypsibiidae by Gąsiorek et. al. [8]. We identified the presence of eight transverse lines of dorsal spines; however, we could not assign the specimens to Calohypsibius ornatus sensu stricto, because this taxon has a high intraspecific variability. The presence of several species has been suggested under this name but there is no detailed redescription [65,8]. Calohypsibius ornatus sensu lato has been reported from North America, in Greenland, Canada, and USA [66], and from South America, in Bolivia, Argentina, and Colombia [67].
Material examined: IV (1) and X (1). Remarks: The specimens correspond well to the key proposed by Fontoura and Pilato [41]. Diphascon mitrense belongs to the pingue-species group, which is a complex of 10 morphologically homogeneous species [41].
This species has been only recorded in Argentina [67]; therefore, the specimens described in this study is the first record from Mexico and North America.
Diphascon pingue (Marcus, 1936) [70]  Remarks: It is believed that Diphascon pingue sensu lato has a cosmopolitan distribution [71]. This species has been reported from North America in Greenland, Canada, and USA [66]; from Central America in Costa Rica [72]; and from South America, in Argentina, Bolivia, Brazil, and Ecuador [67]. From Mexico, it has been recently reported in temperate mountains of Sierra Madre Oriental [15].
Subfamily: Hypsibiinae Pilato, 1969 [63]. Genus: Hypsibius Ehrenberg, 1848 [73]. Hypsibius dujardini sensu lato is a species group [44] with an apparent global distribution [71]; it has been reported from North America, in Canada, Greenland, and USA [66]; from Central America in Costa Rica [72]; and from South America, in Argentina, Bolivia, Chile and Uruguay [67]. On the other hand, Hys. exemplaris has only been recorded from England [44]. Remarks: All collected specimens showed a short claw base and therefore the primary branches seemed to be attached closely to the base of the secondary claw ( Figure S3i), characters that allow to differentiate this species from Hypsibius pallidus Thulin, 1911, (amended by Kaczmarek and Michalczyk, 2009) [42]. However, they differ in the ranges of other body measurements (body length, buccal tube length, macroplaoid 1 length, and base and branches of claw 3) provided in the redescription of Hyspsibius microps Thulin, 1928 (amended by Kaczmarek and Michalczyk, 2009) [42]. Additionally, we observed presence of the minute dot-like septula at the end of the placoids row ( Figure S3h). A robust morphometric analysis is necessary to evaluate the morphological variation of this species and confirm the specific identity of the specimens.
Hypsibius microps is a member of convergens species group [42,44]. This species sensu lato is largely Holarctic [71] and has been recorded from North America, in Canada, Greenland, and USA [66]; from Central America in Costa Rica [72]; and from South America, in Argentina, Brazil and Uruguay [67]. Remarks: All collected specimens show a long claw base and the primary branches seem to be connected at a high level of the secondary claw ( Figure S3l), characters that allow to differentiate this species from Hys. microps [42]. However, they differ in the ranges of other body measurements (body length, macroplacoid 1 length, macroplacoid row length, and base and branches of claw 1 and 4) provided in the redescription of this species by the same authors. Additionally, we observed presence of the minute dot-like septula at the end of the placoids row ( Figure S3k). A morphometric analysis is necessary to evaluate the morphological variation of this species and confirm the specific identity of the specimens.
Hypsibius pallidus is a member of convergens species group [42,44]; this species sensu lato has been reported from North America, in Canada, Greenland, USA and Mexico [11,66]; and from South America, in Argentina, Bolivia and Chile [67].
Subfamily: Itaquasconinae Rudescu, 1964 [74]. Genus: Adropion Pilato, 1987 [34]. Adropion scoticum [75] (Murray, 1905)  Remarks: The collected specimens showed the characters proposed by Murray [75] and Li and Liu [76]. Adropion scoticum sensu lato is a cosmopolitan complex of remarkably similar species, which needs an integrative taxonomic review [66]. Additionally, there is no known type material of Adr. scoticum sensu stricto [66,77]; therefore, a redescription is required. This species has been reported from North America, in Canada, Greenland and USA [66]; from Central America in Costa Rica [72]; and from South America in Argentina, Bolivia, Brazil, Chile, Colombia, Peru, and Uruguay [67]. In this study, we present a new record from Mexico, which expands its distribution into North America.
Material examined: IV (2), XV (1), XLV (2), XLVI (7), XLIX (1), L (1), and LI (10). Remarks: Based on the revision of the hufelandi group by Kaczmarek and Michalczyk [37], two groups of specimens were recognized based on the presence of two types of oral cavity armature (OCA): the maculatus and the patagonicus types, in the maculatus morphotype only the third band of teeth could be observed ( Figure S6b,c), while in the patagonicus morphotype the second and the third bands of teeth can be recognized ( Figure S6f,g). Within both groups, the specimens showed conspicuous morphological differences ( Figure S6a,e), in the sculpture of the cuticle, size of the claws, and presence and ornamentation of lunules ( Figure S6d,h). This suggests the occurrence of more than one species sampled.
Species identification can only be made through observation of the eggs; unfortunately, the eggs found could not be identified at a specific level, due to their poor condition.
Genus: Minibiotus Schuster, 1980 [55]. Remarks: All collected specimens show the characters of Min. sidereus proposed by Pilato et. al. [49]. Cuticle with three types pores ("pearls", sensu Pilato et. al. [49]): circular, elliptical, and starshaped pores ( Figure S7b-d). The largest star-shaped pores are visible on the head and on the legs; prominent large star-shaped pores, with many arms (5-7) are present on each leg of the fourth pair, which should be noted as a specific character ( Figure S7g).
Minibiotus sidereus has only been recorded in Ecuador [49]; therefore, the specimens described in this study are the first record from Mexico and North America.    The specific epithet citlalium is a substantive in genitive that refers to the Náhuatl word citlali, meaning star, due to the presence of star-shaped pores throughout the body. The Náhuatl is an original language spoken by ancient Mexicans in central Mexico from the fifth century AD to present.
Description of Holotype Measurements and statistics are shown in Table 2 (Figure. 4a,b); 83% of the pores correspond to rounded, 10% to multi-lobated (4 tips), and 7% to star-shaped (5-6 tips); the abundance of last two pores types are from 6-10 and from 2-10 respectively ( Table 3). The rounded ones are smaller (0.41-0.86 μm; Figure 5a,b) than the multilobated (0.92-2.38 μm; Figure 5c,d) or star-shaped ones (2.19-3.5 μm; Figure 5e,f). The star-shaped pores are larger at the cephalic and caudal regions of the body, these star-shaped pores are arranged in the cuticle in 11 transverse rows, which become double in the segments of the legs I-III (Figure 4). In the ventral region, the star-shaped pores are less numerous than in the dorsal region and are distributed in eight transversal rows from segment of legs I up to before legs IV (Figure 4c,d).   Table 3. Number of star-shaped pores of four and five tips, the most abundant, on dorsal cuticle of Minibiotus citlalium sp. nov. mounted in polivinil lactofenol medium. The individuals measured correspond to type series (N-number of specimens measured, RANGE refers to the smallest and the largest structure among all measured specimens; and SD-standard deviation). Bucco-pharyngeal apparatus of the Minibiotus type, with five pores around the mouth and ten peribuccal papulae (Figure 6a-c). Oval pharyngeal bulb with triangular apophyses (near to first macroplacoid and about the same size), three macroplacoids and a small microplacoid. Macroplacoid shapes were drop-shaped (m1), granular (m2), and granular or almost quadrangular (m3); macroplacoid length sequence 1 ˃ 2 = 3 ( Figure 2; Table 2). The oral cavity armature is composed of two bands of teeth, which are not visible under PCM and at least the first band of teeth is well visible under SEM (Figure 6d). Legs show no cuticular bars and other thickenings ( Figure 4); each leg with both small and large star-shaped pores on external surface, the larger ones three times larger than the smaller ones ( Figure  3). Fine granulation exclusive on legs IV, on dorsal surface (Figure 7a,b,e) and a large star-shaped pore (5-6 tips) present in each leg (Figure 7c,d), with diameter of 2.4 μm in the holotype. Claws are short and robust, like the hufelandi type with conspicuous accessory points (Figure 7a-f). Lunules under all claws with smooth margins (Figure 7f).  Table 4): freely laid, colorless, spherical, smooth surface (Figure 8a,b) under PCM processes with inconspicuous ornamentation, only visible in few instances; if visible, it showed between three to four annulations (Figure 8a), under SEM processes with four annulations, five in some cases barely visible (Figure 8c); processes longer than width (37-57%), tipped processes flexible (1.53-2.08 μm, Figure 8a,b), and entire base process (Figure 8c). Among the eggs, one from a specimen hatching was observed (Figure 8d). On this individual, the oral apparatus and the star-shaped pores were observed (Figure 8e), as well as a pair of star-shaped pores, one of them conspicuously, on external surface of the legs III (Figure 8f), both diagnostic characters of Minibiotus citlalium sp. nov.  Differential diagnosis. By the presence of star-shaped pores in the cuticle, Minibiotus citlalium sp. nov (Figure 9a) is similar to M. pseudostellarus, M. eichhorni, M. constellatus, M. sidereus and Min. pentannulatus, but differs from the first two species by the presence of eleven transverse rows of star-shaped pores, which are also present in M. constellatus (Figure 9b), M. sidereus (Figure 9c) and Min. pentannulatus (Figure 9d). The star-shaped pores in M. pseudostellarus are randomly distributed [81], and in M. eichhorni are arranged in six transverse rows [82]. The new species differs specifically from M. constellatus, M. sidereus, and M. pentannulatus by two attributes, (1) different macroplacoid sequence 1 ˃ 2 = 3, and (2), each leg (I-III) with two star-shaped pores on external surface, one of them conspicuously larger; the larger ones three times larger than the smaller ones. In M. constellatus the macroplacoid sequence is 1 ˃ 2 ˃ 3, in M. sidereus and M. pentannulatus is 1 ˃ 2 < 3; in M. constellatus each leg with almost two star-shaped pores of similar size, in M. sidereus about three star-shaped pores in each leg, and in M. pentannulatus almost four star-shaped pores in each leg. Additionally, Min. citlalium sp. nov. differs from:

Eggs. (measurements and statistics in
(1) Minibiotus constellatus, only recorded from Peru [51], by absence of granulation on legs I-III (granulation present on all legs in M. constellatus).
(2) Minibiotus sidereus, only recorded from Ecuador [49], by shorter placoid row (5.9-9.0 μm (3) Minibiotus pentannulatus, recorded from Colombia [54] and Tanzania [83] by the pattern of star-shaped pores arranged in 11 transverse rows (double in the segments of the legs I-III) was present in all specimens, which ranged from 142.0-250.0 μm in length, whereas in M. pentannulatus the pattern of star-shaped pores can only be recognized in the smaller animals from 144-203 μm. Minibiotus citlalium sp. nov. also differ from M. pentannulatus by the pt of some claws, as follows: of the claw 1, by lower pt of the external primary branch (11.8-20.7   Stec et. al. [83] registered a population of Minibiotus pentannulatus in Tanzania; these authors add characters to those proposed by Londoño et. al. [54] for M. pentannulatus. These characters also support the differences between this species and M. citlalium sp. nov. These characters are granulation on legs I to IV, differ from M. citlalium sp. nov. which only presents granulation on legs IV (Figure  7a-c); M. pentannulatus presents up to three star-shaped pores of different size, on external surface of legs I-III, the smaller star-shaped pore always present in the centre of the above mentioned granulation patch, meanwhile M. citlalium sp. nov. present a pair of star-shaped pores (one smaller than the other one on external surface of legs I-III), and both pores are above smooth cuticle ( Figure  3); and in M. pentannulatus a cuticular fold with a pore in the centre is present just above the mouth opening and visible well under PCM (only in laterally positioned specimens) and under SEM; meanwhile, in M. citlalium sp. nov. five pores around the mouth are present (Figure 6).
Size effect on morphometric data. Linear regressions of 19 out of 26 continuous body traits of Minibiotus citlalium sp. nov. showed significant correlation with BTL (Supplementary Material Table S1), most of them with r 2 values higher than 0.8. Placoid lengths were uncorrelated with BTL. Of the 19 correlated traits, t-tests from slopes supported that 12 were isometric (slope value not significantly different from 1) and seven were allometric (slope value significantly different from 1) (SM Table S1), the relationship between the trait (Y) and BTL (=body size) (i.e., b-slope) and theoretical values of traits adjusted to mean BTL (Y-intercept) of this species are provided in SM Table S1.

Discussion
As noted in the introduction, the tardigrades of Mexico require more extensive taxonomic studies, and currently, only a few faunistic surveys have been published (e.g., [7,[13][14][15][16][17][18][19][20][21][22]). Most of the previous studies have provided an inventory of tardigrade fauna but from non-systematic samplings. Moreover, the number and size of samples and collection sites were rarely mentioned.
In the present study, from a systematic sampling of 57 moss samples, along an altitudinal gradient, including three moss substrates and four vegetation types, five tardigrade species were recorded (Dip mitrense, Dip. pingue, Adr scoticum, Pil. nodulosus, Min sidereus), three putative species could be associated (i.e., records with insufficient data to complete the identification: Cal cf. ornatus, Hys cf. microps, Hys. cf. pallidus, and Macrobiotus spp.), and a new taxon Min. citlalium sp. nov., was described. For each of these taxa, description and illustrations were provided.
Geographic records Our study confirms the presence of Diphascon pingue and Pilatobius nodulosus species previously recorded in Mexico and provides three new records in the country expanding their distribution in America and in some cases connecting the previous records among North, Central and South America. The presence of Dip. mitrense, Min. sidereus, and Adr. scoticum in Iztaccíhuatl volcano constitute three new Tardigrada records from Mexico, the first two species also new records for North America. Diphascon mitrense and Min. sidereus had only been recorded in their respective type localities, Argentina, and Ecuador [48,49]; therefore, this study provides the second record in America for both taxa, expanding their distribution to North America. On the other hand, Adr. scoticum record connects the previous records in North America [66], with Central and South America [67,72]. Iztaccíhuatl records of Diphascon pingue and Pil. nodulosus demonstrate their occurrence in Mexico, since both taxa have been already recorded from Nuevo León state, [14,15] and in the Sierra Nevada in Popocatépetl volcano [14], respectively.
Additionally, the majority of examined species (Cal. cf. ornatus, Hys. cf. microps, Hys. cf. pallidus) belong to taxonomically difficult groups, or in some cases to species complexes as Hys. dujardini and Dip. pingue. These species groups and complex groups display high levels of morphological intraspecific variation from apparently wide geographic ranges in America and other areas around the world [66,67,72], which makes it difficult to interpret the records of this survey in a geographical context. Further studies considering both morphological and molecular data will clarify the taxonomic identity and distribution of their members.
Tardigrades community. As mentioned above, Popocatépetl and Iztaccíhuatl volcanoes are the main physiographic elements of the Sierra Nevada; they present similar environmental conditions, landscapes, climate, seasonal rain regime, and same vegetation types [24]. Given these common characteristics and geographic proximity of both volcanoes, a similar species composition pattern between Iztaccíhuatl and that recorded previously for Popocatepetl [14] was expected. Although we found an equivalent number of species in both volcanoes, eight in Popocatépetl (Echiniscus kerguelensis, Minesium tardigradum, Macrobiotus echinogenitus, Mac. furcatus, Mac. hufelandi, Ramazottius baumanni, Ram. oberhaeuseri, and Pilatobius nodulosus), and five and three putative species in Iztaccíhuatl volcano, the species composition was dramatically different, because only one species, Pil. nodulosus, was present in both volcanoes. In the present study, Pil. nodulosus was found in moss growing on tree bark and soil, from 3400 to 4000 m asl, across the Abies religiosa and Pinus hartwegii forests. In the Popocatépetl volcano, it was found in the lichen Pseudevernia intensa (Nyl.) Hale and W. L. Culb., growing on bark at 4000 m asl, near the timberline in open Pinus hartwegii forest. This species has already been registered in Pinus and Abies forests, in soil, lichen, leaf litter, and moss growing on bark, and soil in North America [66,71]). Unfortunately, biogeographic or biological comparisons cannot be made because most of the records documented for the Popocatépetl are currently considered doubtful by other authors, as they were supported by a single collection event and the identification was based on the original descriptions available, which in turn was vague and barely detailed [10,14,37,[84][85][86]. Despite that the taxa recorded in the Popocatépetl have been found in different regions of the world [71], recent studies based on morphological and molecular data have shown that they belong to taxonomically difficult species complexes [37,44,[87][88][89].
To adequately compare the tardigrades from both volcanoes, it would be necessary to carry out an appropriate sampling in the Popocatépetl volcano and include both morphological and molecular data. This will allow the clarification of its tardigrade diversity and to propose additional ecological studies that include environmental variables to understand the differences in tardigrade communities, since it is common that tardigrade assemblages display ecological structure across mountain ranges. For example, in the Sierra de Guadarrama (Spain), Guil et. al. [27] found that the richness and abundance of tardigrades were associated with environmental variables at both macro (altitude, vegetation structure, climate, and soil characteristics) and micro (leaf litter type and moss weight) scales.
Systematic considerations. Minibiotus citlalium sp. nov. share with Min. constellatus, Min. sidereus and Min. pentannulatus abundant star-shaped pores in anterior and posterior parts of the body, the distribution pattern of these ornamentations in dorsal cuticle arranged in eleven rows, and similar morphology of eggshell with ringed processes. They also share a holotropical distribution in American and African continent: Min. constellatus recorded in Peru, Min. sidereus in Ecuador and Mexico, and Min. pentannulatus in Colombia and Tanzania. The above characteristics support that these taxa conform a species-group, within which Minibiotus citlalium sp. nov. and Min. sidereus are the most similar in morphology; they present larger stars in the anterior and posterior regions of the body, show a clearly larger pair of stars in the fourth pair of legs, as well as a very similar eggshell. Furthermore, in the present study both species were found in the same samples, so apparently, they also share habitat characteristics. A phylogenetic reconstruction via molecular markers will allow help to clarify their evolutionary relationships.
Allometry in morphometric traits of Min. citlalium sp. nov. In Eutardigrada taxonomy, many continuous traits display correlations with body size [90][91][92]. A considerably proportion of these characters grow proportionally with this trait (i.e., isometric traits), while in others the growth is not proportional (i.e., allometric ones), which in turn makes them unsuitable for taxonomic purposes [93]. As in other Eutardigrada members that display many allometric quantitative traits with respect to BTL [60], our regression analyses of morphometric data from Min. citlalium sp. nov., support that seven traits were allometric and 11 traits were isometric relative to BTL (SM Table S1), indicating that this trend is extensive in eutardigrades.
Ratios (pt indexes) are widely used to eliminate body size effects [33,92,93]; however, their use only successfully eliminate this effects in traits that increase proportionally to body size, but not in those that increase unproportionally (i.e., allometric ones) [59]. To overcome this problem, in this study a protocol [60] that provide parameters from regressions was performed, to obtain the slope (b) and Y intercept (a) in each analyzed trait. This in turn will allow to perform Thorpe normalization and to obtain size-effect free traits, independently of their correlation trend respect to body size (isometric or allometric).

Conclusions
Based on a systematic sampling across a multi-habitat gradient in a temperate mountain of the Trans Mexican Volcanic Belt, we found five tardigrade species, three putative species, one record to genus level, and a new species Min. citlalium sp. nov., which we described. Of them, three are new records for Mexico: Dip. mitrense, Adr. scoticum and Min. sidereus (the first two new for North America). This raises the current number of tardigrade taxa in Mexico from 56 to 61, and in the temperate mountains of the Trans Mexican Volcanic Belt from 8 to 13.