Aspidoscelis costatus costatus (Squamata, Teiidae): high elevation clutch production for a population of whiptail lizards

Clutch size and number of clutches per reproductive cycle are important life history traits that can be influenced by anatomical, physiological, evolutionary, and ecological factors. This report on the clutch size and number of clutches of an endemic Mexican whiptail lizard, Aspidoscelis costatus costatus (Cope, 1878), is based on a study of population at an unusually high elevation for a member of this genus. The study site is located in Ixtapan de la Sal, southeastern Estado de México, Central Mexico, at 2090 m a.s.l. Lizards were sampled in June 2006, and from May to July 2007, where females of Aspidoscelis costatus costatus were collected by hand along a drift fence. Female reproductive condition was evaluated based on abdominal palpation for presence of developing eggs; clutch size was determined by actual counts of either vitellogenic follicles or oviductal eggs. The smallest reproductive female was 77 mm snout-vent length; females produced a minimum of two clutches during the breeding season, the mean clutch size of 6.5 eggs (n = 33) was one of the largest reported for the genus. However, both length and width of its eggs, and the relative clutch mass have not been diminished by development of a large clutch. Additionally, comparisons of clutch size were undertaken within the polytypic A. costatus complex, within the genus Aspidoscelis, and between certain genera of whiptail lizards. This apparently represents the first study of whiptail lizards (genus Aspidoscelis), assessing the aforementioned reproductive characteristics, in a population above 2000 m.


Introduction
Knowledge of the reproductive potential of a population is fundamental to understanding its life history (Stearns 1989) and ecological status. Thus, determination of clutch size and number of clutches per activity cycle are the primary steps in assessing reproductive output. Several factors may act as potential sources of variation in clutch size including ecological (Horváthová et al. 2013), evolutionary (Suárez-Rodríguez et al. 2018;Taylor et al. 2006), anatomical (Suárez-Varón et al. 2019;Taylor et al. 2006), and physiological factors (Méndez-de la Cruz et al. 1993). In general, Aspidoscelis lizards are considered to be wide foragers in search of their prey (Paulissen 1987;Utsumi et al. 2020). Due to their active foraging strategy, body shape, and escape strategies, species in the genus Aspidoscelis tend to show a relatively small clutch size, proportional to their body mass (Vitt and Breitenbach 1993). Previous studies have shown that both clutch size and number of clutches per cycle may vary in a species based on latitude or elevation (McCoy and Hoddenbach 1966;Dixon et al. 1971;Taylor et al. 1992); however, this does not apply to all Squamata species (Fitch 1985;Meiri et al. 2013).
Although Western Mexican Whiptail [Aspidoscelis costatus (Cope, 1878)] is currently treated as a polytypic species (Reeder et al. 2002) with eight subspecies, it is generally understood that it is actually a non-monophyletic species complex of several species including Aspidoscelis costatus costatus (Cope, 1878) = Balsas Basin Whiptail [Reeder et al. 2002 (diversity in the genus); Tucker et al. 2016 (nomenclature based on proof of masculine rather than feminine gender of the generic name); Barley et al. 2019 (diversity in the sexlineatus species group in México)]. Based on Tucker et al. (2016) the name A. costatus costatus replaces A. costata costata (sensu Reeder et al. 2002). This species inhabits various ecological habitats and elevations in the states of Mexico, Guerrero, Morelos, Puebla, Tlaxcala, and Oaxaca (Maslin and Secoy 1986;Gómez-Benitez et al. 2016;Méndez de la Cruz et al. 2018;Barley et al. 2019). Despite the ecological and evolutionary constraints (i. e., body shape, SVL, foraging mode, predator escape tactics) on clutch size within the genus Aspidoscelis (Vitt and Price 1982;Taylor et al. 2006), and although several congeners grow to a much larger SVL [i. e., Aspidoscelis stictogrammus (Burger, 1950), Aspidoscelis sacki (Wiegmann, 1834) and Aspidoscelis costatus occidentalis (Gadow, 1906)], A. costatus costatus shows the largest clutch size reported to date (7.7 eggs, range 4-14; López-Moreno et al. 2016). Our study reports the clutch size and number of clutches in a differ-ent unique high-elevation population (> 2000 m) of Balsas Basin Whiptail (Fig. 1), which is also among the largest clutch sizes in a population of the genus Aspidoscelis.

Methods
The study site is located at Ixtapan de la Sal, southeastern Estado de México, north of the Río Balsas Basin, in Central Mexico (18°50'30"N, 99°39'0"W), at an altitude of 2090 m a.s.l. (Fig. 2), which is considered an unusually high elevation for teiid lizards (Vitt and Breitenbach 1993). Vegetation at the locality consisted of coniferous forest interspersed with tropical deciduous forest and grassland (Fig. 3). The climate is semi-humid and semiwarm with summer rains which typically occur from mid-June through mid-September with annual variation (Hernández-Gallegos and Domínguez-Vega 2012).
Females of A. costatus costatus were captured by hand along a drift fence during their activity period (09:00-17:00 h) in June 2006, and from May to July 2007. The reproductive condition of each adult female was evaluated based on an abdominal palpation and a visual assessment, where the vitellogenic/gravid females showed an expanded contour in the abdomen region (Suárez-Varón et al. 2019). Snout-vent length (SVL) and mass were recorded to the nearest 1 mm and 0.1 g, respectively. Only vitellogenic and gravid females were euthanized via an intraperitoneal injection of sodium pentobarbital, and ovaries and oviducts were removed and placed in 10% neutral buffered formalin. Clutch size was estimated by counting vitellogenic follicles (≥ 3 mm, precision 0.01 mm, López-Moreno et al. 2016) or shelled oviductal eggs when present. We also calculated the relative clutch mass (RCM, based only on oviductal eggs) by dividing clutch mass (precision 0.0001 g) by female´s total mass (including clutch weight; Tinkle 1972). All measurements were carried out following appropriate guidelines for Scientific Animal Use, unnecessary stress was avoided, and individuals were humanely sacrificed. Females were deposited in the Laboratorio de Herpetología, Facultad de Ciencias, Universidad Autónoma del Estado de México, México (voucher numbers pending). All lizards were collected under the Scientific Collector Permit, FAUT 0186 SEMARNAT (Secretaría de Medio Ambiente y Recursos Naturales).
We evaluated the differences between slopes and intercepts of regression lines of vitellogenic follicles and number of oviductal eggs against SVL. The relationship of clutch size and SVL of females was evaluated by Pearson's correlation coefficient. Moreover, we evaluated the differences (using a Student's t test) of clutch size, size of eggs and RCM of A. costatus costatus with other species of Aspidoscelis using data from literature. All data were tested for normality via a Kolmogorov-Smirnov test. Analyses were performed in STATGRAPHICS Centurion XV.II, and results were deemed significant if p < 0.05.

Discussion
Aspidoscelis is the most speciose genus (43 species; Uetz and Hõsek 2020) within the family Teiidae. This genus is divided into five clades (Reeder et al. 2002) and shows a low diversity (Vitt and Breitenbach 1993) (Walker et al. 2003;Walker 2008aWalker , b, 2010. However, egg sizes for these forms do not differ significantly (Table 1). Clutch sizes are larger in cold regions , however, whether these clutch differences are reflective of different ecologies or indicators of species divergence is a pending question. It is well to remember that populations presently treated as A. costatus may include two or more closely related species (Reeder et al. 2002;Barley et al. 2019), though no instances of syntopy between these putative species are believed to occur.
Both ecological and evolutionary factors may adjust the reproductive output in lizards (Suárez-Rodríguez et al. 2018;Suárez-Varón et al. 2019). In general, lizards have clutch sizes that vary with the female´s size, foraging mode or predator escape tactics (Vitt and Price 1982). In species of the genus Aspidoscelis, small clutch size is a general tendency (Dunham and Miles 1985;Vitt and Breitenbach 1993;Hernández-Gallegos 2004;Meiri 2018). However, the mean of 6.5 eggs found in A. costatus costatus: (1) is larger than the mean clutch size in the genus Aspidoscelis, and (2) represents one of the largest clutch sizes reported for this genus (including both gonochoristic and parthenogenetic lineages; Meiri 2018). It is noteworthy that females of A. costatus costatus have a similar mean clutch size (6.5) and a smaller mean SVL (91.2 mm) compared with the southern Mexican congener A. communis (Cope, 1878) (mean clutch size 6.6 eggs, mean SVL 96.5, Walker 1982), and a larger clutch size than the largest females in the genus, A. sacki (mean clutch size 5.    Walker et al. 2003;Walker 2008aWalker , b, 2010). An additional difference between populations is the number of clutches per cycle, multiple in Ixtapan de la Sal (as in A. costatus barrancarum, Walker et al. 2003 (Fitch 1985;Meiri et al. 2013). The elongated body shape (typical of Aspidoscelis lizards) is one constraint on clutch size in the genus, but there may also be an added phylogenetic constraint in certain species (see Taylor et al. 2006;Barley et al. 2019). However, also to be considered are the costs associated with carrying a large clutch in active foragers. This could restrain clutch size in a gravid female of Aspidoscelis through natural selection, since the ability of a lizard to carry "extra" mass would directly affect both the probability of escape from a predator (Vitt and Congdon 1978;Huey and Pianka 1981) and success in foraging. In this sense, as previously recorded in A. costatus costatus from Tonatico , an increase of clutch size in A. costatus costatus at Ixtapan de la Sal, has not affected the size of its eggs (Table 1), or its RCM which did not differ from the RCM average within the genus Aspidoscelis (Mesquita et al. 2016) and is near to A. costatus costatus from Tonatico (0.19;López-Moreno et al. 2016).
The anatomical appellation "whiptail lizards" not only refers to individuals of the genus Aspidoscelis, distributed in North and Central America, but also to lizards of the teiid genera Ameivula, Aurivela, Cnemidophorus, Contomastix, and Glaucomastix (Goicoechea et al. 2016); Cnemidophorus is distributed in Central and South America and the other four genera are restricted to South America. Although details of reproduction differ among whiptail species of these genera, the variation is within the evolutionary and ecological limits provided by oviparity (i. e., all species), lack of parental care (i. e., all species), and clutch frequencies controlled by seasonality (i. e., A. costatus costatus, this study) or not (i. e., Cnemidophorus murinus = C. ruthveni, Burt, 1935;Dearing and Shall 1994) as related to latitude and/or altitude. Variation in clutch sizes is both dependent (i. e., A. costatus costatus) and independent of body size (i. e, C. murinus = C. ruthveni, Dearing and Schall 1994). The species of this study, A. costatus costatus from Ixtapan de la Sal, Estado de México, México, is an example of a whiptail representative of its genus which occurs in the subtropics at a highest known altitude for a reproductive study in the genus. Its reproductive cycle is expressed within seasonal constraints, females are of moderately large reproductive size of 77-111 mm SVL, they produce a relatively large mean clutch of 6.5 ± 1.8 eggs which is correlated with SVL. Compared with a whiptail species of another teiid genus in a coastal area of the state of Ceará, Brazil, namely Ameivula ocellifera (Spix, 1825) (Harvey et al. 2012), Zanchi-Silva et al. (2014) found that adult females produce multiple clutches extending throughout the year, but peaking at the end of the rainy season, has a mean clutch size of only 1.98 ± 0.56 eggs that is positively associated with female body size. However, Sales and Ferire (2016) reported no correlation between clutch size and SVL in A. ocellifera, a difference that requires clarification as to whether it represents accidents of sampling, geographic variation, or a taxonomic causation.
Described as having peculiar reproduction, the whiptail lizard of Bonaire Island, Netherland Antilles, South America, was referenced as C. murinus by Dearing and Schall (1994). It is here considered to be the most divergent example of reproduction compared with a form such as A. costatus costatus. However, Ugueto and Harvey (2010) elevated this insular population to species status using an extant subspecific name, with newly recognized C. ruthveni now considered to be restricted to Bonaire and Klein Bonaire islands and Cnemidophorus murinus (Laurenti, 1768) being restricted to Curaçao and Klein Curaçao islands. Unlike most whiptail lizard species which mainly consume arthropods, as is true of A. costatus costatus (Muñoz-Manzano 2010), both C. ruthveni and C. murinus are not only herbivorous, they exist in very dense populations on their respective islands. Dearing and Schall (1994) reported that females of the Bonaire Island whiptail species (maximum SVL of 116 mm), C. ruthveni sensu Ugueto and Harvey (2010), "…typically [produce a clutch of] one very large egg, but some females may produce two eggs." Also, that some insular lizards may produce smaller clutches which produce larger hatchlings (than closely related mainland species of similar size) is a very well-known phenomenon called the island syndrome .
Although data collected suggest a high reproductive output in A. costatus costatus at Ixtapan de la Sal, there are multiple variables that should still be investigated such as dorsal coloration, interlimb distance, and coelomic volume. These future studies will provide a holistic view of reproduction to see if the females in our population have adopted atypical strategies (i. e., seasonal dorsal coloration and widening of the abdominal area), which help to maintain a high-reproductive output as in A. costatus costatus from Tonatico . Finally, further studies on other populations are needed to clarify the idea that probably a large clutch size is characteristic of Balsas Basin Whiptail (A. costatus costatus), which may represent a local adaptation to high predation regimes on their eggs and/ or to low survivorship rates .