Zoodiversity_06_2021.indb UDC UDC 598.11:591.156(212.7:82) CONTRIBUTIONS TO THE KNOWLEDGE OF SEXUAL DIMORPHISM IN LIOLAEMUS DARWINII (SQUAMATA, LIOLAEMIDAE) IN THE MONTE DESERT OF ARGENTINA G. N. Castillo1,2,3*, C. J. Gonzalez-Rivas4 & J. C. Acosta1,3 1Facultad de Ciencias Exactas, Físicas y Naturales, Universidad Nacional de San Juan, Av. Ignacio de la Roza 590, 5402, San Juan, Argentina 2Becario de CONICET (Consejo Nacional de Investigaciones Científi cas y Técnicas), Av. Ignacio de la Roza 590, 5402, San Juan, Argentina 3Gabinete de investigación DIBIOVA (Diversidad y Biología de Vertebrados del Árido), Universidad Nacional de San Juan, Av. Ignacio de la Roza 590, 5402, San Juan, Argentina 4Centro de Rehabilitación de Fauna Silvestre, Educación Ambiental y Recreación Responsable, San Juan, Argentina *Corresponding author E-mail: nataliocastillo@gmail.com Contributions to the Knowledge of Sexual Dimorphism in Liolaemus darwinii (Squamata, Liolaemidae) in the Monte Desert of Argentina. Castillo, G. N., Gonzalez-Rivas, C. J., Acosta, J. C. —Th e lizard Liolaemus darwinii (Liolaemidae) is a typical species with wide distribution in Monte environments of Argentina. Th e objective of this study is contribute to knowledge of sexual dimorphism in a population of L. darwinii (Bell, 1843). We evaluated sexual shape variation of the cephalic region through procustes analyses with geometric morphometry. We predict that the heads in males will have diff erences in shape with respect to females. Th e results showed signifi cant sexual diff erences in shape, mainly in the region around the eyes. Th ere were no signifi cant diff erences in sizes. Geometric morphometry analyses are a useful tool for addressing sexual diff erences in Monte lizards. Th is constitutes the fi rst study for the center-west of Argentina in San Juan province that implements these geometric morphometry analyses. K e y w o r d s : Argentina, San Juan, Monte, Liolaemus darwinii, geometric morphometry. Introduction Reptiles are important in dimorphism research, due to the considerable variety of dimorphisms and life histories observed within this group (Cox et al., 2007). One aspect analyzed in dimorphism is size, although the diff erence between male and female adults is very common in organisms with separate sexes (Fairbairn, 2007). In addition, in most lizards, males tend to be larger than females (Cox et al., 2007; Cabrera et al., 2013). Diff erent hypotheses have been proposed to explain this phenomenon, associated with sexual, natural (ecologi- cal interactions) and fertility selection (Hedrick & Temeles, 1989; Fairbairn, 1997; Cox et al., 2007; Vincent & Herrel, 2007). Th e problem is that these mechanisms lead to the same phenomenon and can be problematic (Anderson & Vitt, 1990; Pincheira-Donoso, 2012). Zoodiversity, 55(6): 479–484, 2021 DOI 10.15407/zoo2021.06.479 480 G. N. Castillo, C. J. Gonzalez-Rivas, J. C. Acosta Another interesting aspect to analyze is the variation in shape between sexes (Benitez, 2013). So far, shape variation with geometric morphometry in lizards has been little explored in Argentina, only with contribu- tions of Minoli et al. (2016) and González-Marín et al. (2016). Geometric morphometry (GM) is a statistical approach to study shape variation and its co-variation with other variables, it allows visualizing both the size and direction of shape change among groups. Th is methodology has considerable power to detect small diff er- ences in shape. Such shapes refer to the geometric properties of an object that do not vary with changes in its location, rotation or size (Aguirre & Prado, 2018). GM is based in landmarks with coordinates in two or three dimensions defi ned in pictures or scans. In GM, landmarks are used to defi ne the shape, and identify the same anatomical point in all the individuals of the study. Th us, landmarks with coordinates allow describing such variation keeping the shape of the geometric structure intact (Aguirre & Prado, 2018). Geometric morphometry is a useful tool to detect diff erences among reptiles species, as well as shape variation among sexes (González-Marín et al., 2016; Minoli et al., 2016; Murta-Fonseca et al., 2019). Its frequent use is due to Geometric morphometry helps to elucidate diff erences among species with a poor morphological variation or scarce sexual variation among individuals (González-Marín et al., 2016). Liolaemus darwinii (Bell, 1843) is a lizard species distributed in many provinces of Argentina (Abdala et al., 2012), with a mean size of 65 mm (Cei, 1986; Acosta et al., 2017). It is a typical Monte form to the point that it is almost an indicator (Cei, 1986). It is an oviparous species, with two layings per year and a mean clutch size of 3–6 eggs (Cei, 1986; Acosta et al., 2017). Its conservation status is non-threatened (Abdala et al., 2012). To date, morphometric studies with GM have not been carried out in L. darwinii, although data on the size of the head with lineal morphometry have been reported (Cabrera et al., 2013). Due to this, the objective is to analyze the shape variation in L. darwinii’s head through geometric morphology. We analyzed the cephalic region of 30 adult specimens of L. darwinii (16 females, 14 males) (fi g. 1). Lizard were captured at El Encón locality, Department 25 de mayo, San Juan province (32°12' S, 67°47' W) (fi g. 2). Th is sector is represented by the phytogeographic province of Monte, covering extensive arid areas with an average rainfall of less than 100 mm / year, with years without any records. It covers an approximate area of 40,499 km2, corresponding to 45 % of the total province. Predominantly xerophytic plants adapted to a warm and dry climate, with little summer rainfall. Vegetation responds to wet and dry cycles. It is characterized by the presence of shrub steppe exceeding 3 m high, which branch from the base (Morello, 1958). Sampling was conducted dur- ing 3 years 2017, 2018 and 2019. 16  pit fall  traps  were  placed  random- ly  (width  25  cm,  height  37  cm). Th e traps were buried at a depth of 37 cm. Captured individuals were sacrifi ced by intraperitoneal administration of sodium thiopental, fi xed with 10 % formalin and preserved in 70 % alcohol. All the specimens were deposited in the Herpetological Collection, Department of Biology, Faculty of Exact, Physical and Natu- ral Sciences of the National University of San Juan (Liolaemus darwinii: UNSJ, 4033–4052). Image captures were taken with a Nikon® COOLPIX P520 42x digital camera. Pictures of all the specimens were taken inside a light box (Minoli et al., 2016). Th e photo- graphic camera was mounted on a tripod with the same focal distance length (40 cm), which remained constant in each photography. Th e dorsal view of the head of each lizard was cap- tured, and the scale was recorded with a grid. Subsequently, images were downloaded in a computer and a tps. fi le was created using tpsUtil 1.58 ( Rohlf, 2013 b). Reference points were selected to quantify any detectable dif- ference between lizards, to meet the following assumptions: homology, cover, repeatability, consistency in the relative position and copla- narity (Toro-Ibacache et al., 2010). Eight Type 1 landmarks (landmarks in discrete points formed by the intersection of Fig. 1. Female specimen of L. darwinii. Fig. 2. Sampling area, locality of the Encón, dpto. May 25, province of San Juan, Argentina. 481Contributions to the Knowledge of Sexual Dimorphism in Liolaemus darwinii… tissues) of adult specimens were included, and all the reference points were digitized on the right dorsal side of the head for each sample (fi g. 3) using tpsDig v. 2.17 (Rohlf, 2013 a). Lizards showing anomalies (n= 10) (e. g., lumps, scars, bites, twisted head) were exclud- ed, since in those cases landmarks could not be accurately established (Minoli et al., 2016). Pictures were taken in the instant euthanasia was carried out. To avoid the precision error, all the photomontages were performed by the same person. Th e consensus shapes, partial wraps and relative wraps were generated with the tpsRelw soft ware version 1.69. Using the MorphoJ soft ware v. 1.06 (Klin- genberg, 2011), a generalized procustes analysis was carried out. Th is allowed the elimination of the individual variation components (in imag- es) that did not correspond to the shape. Land- mark alienation of all the individuals under study was carried out, in such way that the two- dimensional coordinates (x, y) represent shape variation (Aguirre & Prado, 2018). To model how shape varied between specimens, defor- mation grids were used. Additionally, an outlier test was performed to control and exclude liz- ards that widely diverted from the mean. To explore shape variation patterns identifying the directions of higher variance, principal component analyses were carried out. Th e information on size was retained as centroid size (CS). To explore size variations, an ANOVA with centroid size as dependent variable, and sex (male/female) as indepen- dent variable was carried out. For a better visualization of the data, a boxplot diagram with the centroid size was performed. A regression test between the Procustes coordinate data (which refl ect individuals shape) and centroid size was carried out to correct the diff erences related with allometry. “Procustes coordinates” was selected as the dependent variable, and “Log Centroid Size” as the independent variable. A “Perform per- mutation test” was carried out with 10.000 permutations. Th e CVA (Canonical Variates Analysis) analysis was performed, and “Re- siduals, Regression” was selected to carry out the analysis independently of allometry. Results Th e principal components 1 (28  %), 2 (20  %) and 3 (17  %) were the axes with the highest variation in the new space, explaining 65 % of the original variance associated to female and male variation (table 1) (fi g. 4). Diff erences in the cephalic region shape between L. darwi- nii males and females were found Fig. 3. Landmark type 1 in L. darwinii. Cephalic scales (1): Pp — postparietal (2); P — pineal (3); In — internasal, (4); N — nasal (5, 6, 7, 8); SpO — supraocular. Females and males of L. darwinii, shape comparison. Deformation grids, showing the sectors where the shape diff ers most between sexes. Cross- validation scores: Th e residual form of the regression was used to assess sexual dimorphism (corrected size) by discriminant analysis. Th e diff erence in shapes between females and males of L. darwinii is observed in the dorsal cephalic region. Fisher’s discriminant rule (x-axis), with the cut-off point at a value zero: males with positive values and females with negative values. T a b l e 1 . Main components for the variation of shape in L. darwinii Autovalues Variance, % Accumulation, % 1 0.00119 28.03 28 2 0.00086 20.184 48 3 0.000759 17.812 66 4 0.000442 10.381 76 5 0.000353 8.284 84.69 6 0.000239 5.607 90 7 0.000108 3.143 93.4 8 0.0000782 2.536 95.9 Fig. 4. Morphological separation of the cephalic region of fe- males and males using main components. 482 G. N. Castillo, C. J. Gonzalez-Rivas, J. C. Acosta (ANOVA, Procustes F = 2.29; df = 12; p = 0.01) (table 2). Deformations in the grids, supraocular scales (land- marks 5, 6, 7, 8) were observed. Such deformation regions corresponded to the sector where confi guration dif- fered the most (fi g. 3). Males showed a laterally more widened shape, ob- served in landmarks 1, 2, 7 and 8; as well as higher longitudinal arrange- ment. In fi gure 3, the cross-validation analysis is observed (cross-validation scores), showing shape diff erences in the cephalic region between males and females of L. darwinii. An over- lap between males and females can be seen, determining subtle although ex- istent diff erences in shape (fi gs 3 and 4). Males and females did not show signifi cant diff erences considering centroid size, although males present- ed higher sizes (table 3) (fi g. 5). Discussion and conclusion Morphological sexual variations (sexual dimorphism) act in a diff er- ential way in males and females, and they are, thus, the evolutionary result of selective pressures (Pianka, 1982). To date, three mechanisms have been proposed that could explain the evo- lution of sexual dimorphism (Cox et al., 2007): one based on sexual selec- tion mechanisms (i. e., males have competition advantages); a second one based on intersexual competition related to resource use (i. e., ecological interactions); and a third one based on reproductive roles, named by Fairbairn et al. (2007) as “fertility selection”, which favors the size of larger females. However, several phenomena could lead to the same result. In the case of sexual selection and ecological interactions may interact synergistically during the evolution of sexual dimorphism (Bolnick & Doebeli, 2003). Our results indicated that L. darwinii presented head shape variation, specifi cally ex- hibited statistical diff erences in the shape of the eye orbit. Th ese correspond to supraocular scales, indicated by landmarks 5, 6, 7 and 8. To date, in L. darwinii there was only head information based on linear morphology, with the head of males larger than females (Ca- brera et al., 2013). Sexual dimorphism with linear morphology has been frequently evalu- ated for other species of the desert of the mount indicating similar results such as L. olon- gasta Etheridge, 1993, L. riojanus Cei, 1979, L. cuyanus Cei & Scolaro, 1980 and L. acostai (= L. pseudoanomalus) Abdala & Juárez-Heredia, 2013 (Villavicencio et al., 2003; Cánovas et al., 2006; Laspiur et al., 2006; Laspiur & Acosta, 2007). T a b l e 2. Generalized analysis of Procrustes, ANOVA, for shape variation. sexual, individual variation and measurement error is observed Eff ect SS MS df F p Sexes 0.0164 0.001 12 2.19 0.01 Individual 0.2175 0.0006 348 8.44 0.0001 Error 0.02 0.00007 348   T a b l e 3. Generalized analysis of Procrustes, ANOVA. Sexual variation in size, individual and measurement error are observed Eff ect SS MS df F p Extra 1 2948.7 2948.77 1 0.16 0.6 Individual 544670.31 18781.734 29 20.07 0.0001 Error 1 27137.224 935.766 29     N o t e . Extra 1 — variation between sexes; individu- al — individual variation. Fig. 5. Variation in size of the cephalic region between males and females of L. darwinii, where it is observed an absence of signifi cant diff erences between both sexes. 483Contributions to the Knowledge of Sexual Dimorphism in Liolaemus darwinii… With regard to the diff erence in shape in L. darwinii, Vidal et al. (2011) analyzed the eye orbit in Liolaemus tenuis and found signifi cant diff erences between sexes. Males have a more rounded and extended orbit shape than females. Th ese authors suggested that they correspond to a potentially adaptive character associated with social condition (Vidal et al., 2011). In our case study in L. darwinii, to date, we do not have relevant information to associate it to a social system. However, the shape of the orbit could be related to a polygy- nous social system in reptiles (Vidal et al., 2011). In addition, Vidal et al. (2005) mention that morphological diff erences in eye orbits are probably also related to territorial defense. Regarding our results, we believe that the shape diff erences found between sexes in L. darwinii could be related to sexual selection. Sexual selection is related in a context where male size confers an advantage in male-male competition (Cox et al., 2007). Th at is, body size determines success in agonistic encounters between males and territoriality (Shine et al., 1989; Cox et al., 2007). Territorial species show a marked dimorphism in size (Cox et al., 2007). Shine (1989) mentions other types of sexual interactions; exhibition and courtship, forced insemination, couple transport and provision of nuptial gift s between males and fe- males. Although not necessarily these types of interactions occur in reptiles. Regarding the ecological hypothesis for sexual dimorphism (Cox et al., 2007), an ecological eff ect on dimor- phism is likely when it occurs in a character that is free from sexual selection (Shine, 1989; Bolnick & Doebeli, 2003). Th ere is evidence that morphological dimorphism is not related to trophic resource (Shine, 1989). For other mount species in Argentina, it has been reported that the largest head size in males, might be related to aggressive interactions between males for access to females and territory defense (Cánovas et al., 2006). Th us, sexual selection could favor large size in males (due to combat between males) but not in females (Shine, 1989). We emphasize that all three processes; selection of fertility, sexual and ecological di- vergence can operate in the same population. Size can be a consequence of any of these factors acting alone or together (Shine, 1989). As a conclusion, sexual dimorphism is a common phenomenon in lizards, and it is frequent in Liolaemus spp. (Valdecantos & Lobo, 2007; Cabrera et al. 2013). Th us, in our study, females and males of L. darwinii showed a diff erential adaptation to the environment, and consequently, shape variation in males and females could interact diff erentially in their habitat. In summary, the utility of geometric morphometry as a quantitative tool to diff erenti- ate sexes in L. darwinii is observed. Geometric morphometry analysis is an excellent tool to distinguish sexes and can be complemented with lineal morphometry studies. We thank the Sub-Secretary of the Environment for the permits granted (Nº 1300-3097-16), and the rangers (Mariano Hidalgo, Jorge Cayuela, Jesús Quiroga and José Castro) for their help in fi eld samplings. Sofía Nanni assisted us in draft ing the English version. We thank two anonymous reviewers for improving the manuscript. Th e authors declare no competing interests. References Abdala, C. S., Acosta, J. C, Acosta, J. L., Álvarez, B. B., Arias, F., Ávila, L., Blanco, M. G., Bonino, M. J., Bo- retto, M., Brancatelli, G., Breitman, M. F., Cabrera, M., Cairo, R. S., Corbalán, V., Hernando, A., Ibar- guengoytía, N. R., Kacoliris, F., Laspiur, A., Montero, R., Morando, M., Pelegrín, N., Pérez, C. H. F., Quinteros, A., Semhan, S. R., Tedesco, M. E., Vega, L., Zalba, S. M. 2012. Categorización del estado de conservación de las lagartijas y anfi sbenas de la República Argentina.  Cuadernos de Herpetología, 26, 215–248. http://sedici.unlp.edu.ar/handle/10915/25472 Acosta, J. C., Blanco, G. M., Gómez-Alés, R., Acosta, R., Piaggio-Kokot, L., Victorica, A., Villavicencio, J., Fava, G A. 2017. Los reptiles de San Juan. 1ª edición, editorial Universidad Nacional de San Juan (UNSJ). San Juan, Argentina. Aguirre, W., Prado, J. P. 2018. Guía práctica de Morfometría Geométrica. Aplicaciones en la Ictiología. Pontifi cia Universidad Católica del Ecuador Sede Esmeraldas (PUCESE). Anderson, R. A, Vitt, L. J. 1990. Sexuaal selection versus alternative causes of sexual dimorphism in teiid lizards. Oecologia, 84, 145–157. Benitez, H. A. 2013. Sexual Dimorphism Using Geometric Morphometric Approach. In: Moriyama, H, ed. Sex- ual Dimorphism. Rijeka: In Tech, 35–50. Bolnick, D. I., Doebeli, M. 2003. Sexual dimorphism and adaptive speciation: two sides of the same ecological coin. Evolution, 57 (11), 2433–2449. 484 G. N. Castillo, C. J. Gonzalez-Rivas, J. C. Acosta Cabrera, M. P., Scrocchi, G. J., Cruz, F. B. 2013. Sexual size dimorphism and allometry in Liolaemus of the L. lurenti group (Sauria: Liolaemidae): Morphologic lability in a clade of lizards with diff erent reproduc- tive modes. Zoologischer Anzeiger, 252, 299–306. http://dx.doi.org/10.1016/j.jcz.2012.08.003 Cánovas, M. G., Villavicencio, H. J., Acosta, J. C., Marinero, J. A. 2006. Dimorfi smo Sexual y Morfometría de una Población de Liolaemus olongasta (Iguania: Liolaeminae) en la Laja, Albardón, San Juan, República Argentina. Cuadernos de Herpetología, 19, 57–61. http://sedici.unlp.edu.ar/handle/10915/6433 Cei, J. M. 1986. Reptiles del Centro, Centro-Oeste y Sur de la Argentina. Herpetofauna de Zonas Áridas y Semiáridas. Museo Regionale di Scienze Naturali Torino. Monografía IV, 112–120. Cooper, W. E., Vitt L. J. 1989. Sexual dimorphism of head and body size in an iguanid lizard: paradoxical results. American Naturalist, 729–735. https://www.journals.uchicago.edu/doi/abs/10.1086/284948?journalCode=an Cox, R. M., Butler, M. A., John-Alder H. B. 2007. Th e evolution of sexual size dimorphism in reptiles. In: Fair- bairn, D. J., Blanckenhorn, W. U., Székely, T. Sex, size, and gender roles: evolutionary studies of sexual size dimorphism. University Press, Oxford, 38–49. Fairbairn, D. J. 2007. Introduction: the enigma of sexual size dimorphism. In: Fairbairn, D. J., Blanckenhorn, W. U., Széke- ly, T., eds. Sex, size, and gender roles: evolutionary studies of sexual size dimorphism. University Press, Oxford, 1–10. González-Marín, A., Morando, M., Ávila, L. J. 2016. Morfología lineal y geométrica en un grupo de lagarti- jas patagónicas del género Phymaturus (Squamata: Liolaemini).  Revista mexicana de biodiversidad, 87, 399–408. https://doi.org/10.1016/j.rmb.2016.04.009 Hedrick, A. V., Temeles, E. J. 1989. Th e evolution of sexual dimorphism in animals: hypotheses and tests. Trends in Ecology & Evolution, 4 (5), 136–138. Klingenberg, C. P. 2011. MorphoJ: an integrated soft ware package for geometric morphometrics. Molecular Ecology Resources, 11, 353–357. https://doi.org/10.1111/j.1755-0998.2010.02924.x Laspiur, A., Acosta, J. C. 2007. Dimorfi smo Sexual de Liolaemus cuyanus Cei and Scolaro, 1980 (Iguania: Li- olaemidae) en una población de San Juan, Argentina. Revista Peruana de Biología, 14, 47–50. http://www. scielo.org.pe/scielo.php?pid=S1727-99332007000200010&script=sci_arttext&tlng=en Laspiur, A., Ripoll, Y., Acosta J. C. 2006. Dimorfi smo sexual de Liolaemus riojanus (Iguania: Liolaemidae) en una población de un desierto arenoso del Monte de San Juan, Argentina. Revista Española de Herpe- tología, 20, 87–94. https://dialnet.unirioja.es/servlet/articulo?codigo=2342392 Minoli, I., Morando, M., Avila, L. J. 2016. Sexual dimorphism and interspecifi c head variation in the Liolaemus mela- nops complex (Squamata: Liolaemini) based on geometric morphometrics. Th e Herpetological Journal, 26, 225– 240. https://www.ingentaconnect.com/contentone/bhs/thj/2016/00000026/00000003/art00006 Morello, J., 1958. La Provincia Fitogeográfi ca del Monte. Opera Lilloana II, Tucumán. Murta-Fonseca, R. A., Machado, A., Lopes, R. T., Fernandes, D. S. 2019. Sexual dimorphism in Xenodon neu- wiedii skull revealed by geometric morphometrics (Serpentes; Dipsadidae). Amphibia-Reptilia, 40, 461– 474. https://doi.org/10.1163/15685381-20191147 Pianka, E. R. 1982. Ecología evolutiva. Edit. Omega, Barcelona. Pincheira-Donoso, D. 2012. Selección y evolución adaptativa. Fundamentos teóricos y empíricos desde la perspec- tiva de los lagartos. Ediciones Universidad Católica de Chile, Santiago, 1–403. Rohlf, F. J. 2013 a. tpsDig. version 2.17. Department of Ecology and Evolution, State University of New York at Stony Brook, Stony Brook, NY. Rohlf, F. J. 2013 b. tpsUtil, version 1.38. Department of Ecology and Evolution, State University of New York at Stony Brook, Stony Brook, NY. Shine, R. 1989. Ecological causes for the evolution of sexual dimorphism: a review of the evidence. Th e Quar- terly Review of Biology, 64, 419–461 Toro-Ibacache, M. V., Manriquez, S. G., Suazo-Galdames, I. 2010. Morfometría geométrica y el estudio de las formas biológicas: de la morfología descriptiva a la morfología cuantitativa. International Journal of Mor- phology, 28, 977–990. http://dx.doi.org/10.4067/S0717-95022010000400001  Valdecantos, M. S., Lobo, F. 2007. Dimorfi smo sexual en Liolaemus multicolor y L. irregularis (Igua- nia: Liolaemidae).  Revista Española de Herpetología, 21, 55–69. https://dialnet.unirioja.es/servlet/ articulo?codigo=2877437 Valladares, J. P., Etheridge, R., Schulte, J. II., Manriquez, G., Spotorno, A. 2002. Nueva especie de lagartija del norte de Chile, Liolaemus molinai (Reptilia: Liolaeminae). Revista chilena de historia natural, 75, 473–489. http://dx.doi.org/10.4067/S0716-078X2002000300002  Vidal, M. 2011. Eye orbit geometric shape in Liolaemus as an indicator of polygyny or monogamy. Gayana, 75 (2), 155. Vidal, M., Ramírez, C., Ortiz, J. C., Lamborot, M. 2005. Intraspecifi c variation in morphology and sexual di- morphism in Liolaemus tenuis (Tropiduridae). Amphibia–Reptilia, 26 (3), 343–351. Villavicencio, H. J., Acosta, J. C., Cánovas, M. G., Marinero, J. A. 2003. Dimorfi smo sexual de Liolaemus pseu- doanomalus (Iguania: Liolaemidae) en el centro-oeste de Argentina. Revista española de Herpetología, 17, 87–92. http://www.herpetologica.org/revespherp/vol17_2003/Dimorfi smo%20sexual.pdf Vincent, S., Herrel, A. 2007. Functional and ecological correlates of ecologically-based dimorphisms in squa- mate reptiles. Integrative and Comparative Biology, 47, 172–188. 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