JOURNAL OF MORPHOLOGY 277:107–117 (2016) Eye and Pit Size are Inversely Correlated in Crotalinae: Implications for Selection Pressure Relaxation Yang Liu,1 Qin Chen,1 Theodore J. Papenfuss,2 Fang Lu,3* and Yezhong Tang1 1Department of Herpetology, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, China 2Department of Amphibians and Reptiles, Museum of Vertebrate Zoology, University of California, Berkeley, California 3Department of Ophthalmology, West China Hospital, Sichuan University, Chengdu, China ABSTRACT Mate, prey, and predator recognition improved by neural image sharpening mechanisms often depend on the integration of information from in the medulla (Stanford and Schroeder, 1980) and multiple sensory modalities including visual, auditory, by the fact that ascending pathways conveying IR and/or olfactory inputs. In Crotalinae, the eyes sense and visual information converge in the forebrain visible light while the pit organs detect infrared (IR) radiation. Previous studies indicate that there is signif- (Berson and Hartline, 1988). In addition, the IR- icant overlap between the eye and pit sensory fields senses normally work in concert with the visual and that both senses are involved in recognition proc- system in facilitation of prey detection, identifica- esses. This study investigated the relationships tion, and localization (Ford and Burghardt, 1993). between eye and pit sizes in this taxonomic group as a Nevertheless it is still unclear if the IR-system function of phylogeny and habitat. In view of the fact and visual system normally work in parallel since that pit orientation depends largely on snout shape, pit the physical characteristics of the visible (0.38– vipers were grouped as follows: 1) arboreal, 2) terres- 0.75 mm) and IR (0.8–30 mm) radiation emitted by trial with rounded snout, and 3) terrestrial with endotherms are different. pointed snout. The pit orientations and habitant pat- terns were fully independent of the Crotalinae phyloge- It is thought that the evolution of the IR- netic tree. The phylogenetic generalized least squares sensory system has made Crotalinae the most model showed that both eye and pit areas were not of species-rich taxon in the Viperidae, containing significantly phylogenetic relatedness, implying alter- over 200 species (Malhotra and Thorpe, 2004; Cas- natively a strong effect of adaptation on eye and pit toe and Parkinson, 2006; Orlov et al., 2009; David sizes. Negative correlations between relative eye and et al., 2011; Pyron et al., 2013). Pit vipers inhabit pit areas in terrestrial (both pointed and rounded various habitats, with major radiations in both the snouts) and arboreal species were statistically signifi- Old and New Worlds (Zhao et al., 1998; McDiar- cant. Our results suggest that the eyes and pits func- tion in a complementary fashion such that selection for mid et al., 1999). An abundant diversity of pit IR-perception relaxes selection pressures on the visual viper species and ecological patterns provides an system and selection for visual discrimination relaxes excellent opportunity to investigate morphological selection pressures acting on the IR-system. J. Mor- adaptation associated with the evolution of multi- phol. 277:107–117, 2016. VC 2015 Wiley Periodicals, Inc. modal sensory processing. In pit vipers, eye and pit sensory fields largely KEY WORDS: infrared perception; vision; pit character; overlap. Structurally, both visual and IR-inputs phylogeny; adaptation are projected to the pit viper optic tectum where a layer of IR-sensitive neurons is located subjacent to the visual layer (Berson and Hartline, 1988). INTRODUCTION Crotalinae snake species (pit vipers) are unique among animals capable of detecting middle and Contract grant sponsor: National Natural Science Foundation of long wavelength infrared (IR) radiation insofar as China; Grant number: NSFC 31272304 (to Y.T.) and 31301882 (to pit vipers are capable of IR-imaging (Campbell Q.C.); Contract grant sponsor: China Postdoctoral Science Foun- dation; Grant number: PFC 2013M542301 (to Q.C.). et al., 2002). The pit organ located between the eye and nostril on each side of the viper’s face *Correspondence to: Fang Lu; Department of Ophthalmology, serves as an IR-detector. Behaviorally, the facial West China Hospital, Sichuan University, Chengdu, China. pit appears to function as an “eye” because it can E-mail: [email protected] compensate for visual deprivation (Kardong and Mackessy, 1991; Kardong and Berkhoudt, 1999). Received 6 October 2014; Revised 14 August 2015; Pit vipers perceive a visual-thermal multispectral Accepted 2 September 2015. image (Newman and Hartline, 1982; Moiseenkova Published online 7 October 2015 in et al., 2003). This conclusion is supported by the Wiley Online Library (wileyonlinelibrary.com). fact that the resolution of the thermal image is DOI 10.1002/jmor.20483 VC 2015 WILEY PERIODICALS, INC. 108 Y. LIU ET AL. Functionally, the infrared and visual spatiotopic ences with behavioral observations and specimen collection; 2) tectal maps have similar but not identical axes; influential monographic books (Zhao et al., 1998; Zhao, 2006) the IR-magnification is greater than that for clas- and 3) the published literature. In this study, the genera Bothriopsis (Kwiatkowski and sical vision (Hartline et al., 1978). Nevertheless Burt, 2011), Bothriechis (Campbell and Smith, 2000), Trimere- visual and IR-information are projected in register surus (Malhotra and Thorpe, 1997), and Tropidolaemus (Tsai spatially and temporally to the midbrain and the et al., 2012) were considered as arboreal. All other genera were available evidence indicates that the snakes can categorized as terrestrial, and then grouped into two patterns use either modality for hunting prey. based on their snout shapes and pit orientations, which are shown in Table 1. Chen et al. (2012) found that snakes with either Although most pit vipers show no preference for being active the pit organs or eyes occluded on both sides of diurnally or nocturnally, primarily nocturnal foragers were the face exhibit about 75% success in prey capture. selected by searching the snake ecology literature. These Similarly, occlusion of both eyes or both pits con- included Bothropoides jararaca (Sazima, 1992), the genus strict strike angles during prey capture to the Bothrops (Wasko and Sasa, 2009), Calloselasma rhodostoma (York, 1984), Ovophis monticola (Malhotra et al., 2011), same extent, within 308 to the left and right sides Ovophis okinavensis (Mori et al., 2002), Protobothrops mucros- of the midline (Chen et al., 2012). In addition, con- quamatus (Huang et al., 2007), and Trimeresurus stejnegeri genitally blind pit vipers using IR-imaging have (David et al., 2001). been shown to aim and strike at prey as accu- rately as normal siblings under a variety of condi- Construction of Phylogenetic Trees tions (Kardong, 1986; Kardong and Mackessy, The time-calibrated tree of Crotalinae was derived from a 1991). Finally, neurophysiological studies support recent comprehensive phylogenetic tree of 4,161 species of the idea that binocular thermal stereopsis occurs Squamata (Pyron and Burbrink, 2014). The species not in- in the viper midbrain (Goris and Terashima, 1973; cluded in this study were pruned from the analyses with pack- Berson and Hartline, 1988). age “caper” (Orme et al., 2012; Han and Fu, 2013). In view of these studies, we hypothesized that Data Analysis selection favoring IR-perception could relax the selection pressures on development of the visual Phylogenetic generalized least squares regression analysis system because these two modalities can func- (PGLS) was performed in R i386 3.1.2 to test the degree of dependence of the biometric variables on the phylogenetic tion in a complementary and perhaps indepen- covariance (R Development Core Team, 2012). Residuals of the dent way. Thus, we predicted that eye and pit eye and pit areas were calculated in order to minimize allomet- sizes would be inversely correlated across the ric scaling (Revell et al., 2007). A phylogenetic parameter k whole Crotalinae, yet vary independently with (Pagel, 1999) was used as the measure of phylogenetic signals for continuous traits, ranging from 0 (no phylogenetic signal, phylogeny. equivalent to a “star” phylogeny) to 1 (consistent with Brown- ian motion, BM), while intermediate values imply that the data MATERIALS AND METHODS support a model with status between “star” phylogeny and BM. Its optimum value and confidence limits were estimated using Morphological Measurements the “pgls” function in packages “caper” with a maximum likeli- A total of 167 specimens were examined, belonging to all 24 hood method when performing a correlation analysis (Orme genera, including 59 species of Crotalinae (Table 1). The data et al., 2012). The relationship between the eye and pit areas in were obtained from specimens stored in the Museum of Verte- Crotalinae was evaluated using PGLS, applying a covariance brate Zoology, UC Berkeley and the National Museum of Natu- matrix based on Brownian and Ornstein–Uhlenbeck (OU) ral History of the Smithsonian Institution, USA. All specimens motion models of evolution with functions “gls,” “corBrownian” used for size measurement had been fixed in ethanol for pro- (Freckleton et al., 2011), and “corMartin” (Martins and Hansen, longed periods. In order to eliminate the effect of asymmetry on 1997) in packages “nlme” (Abellan and Ribera, 2011), and “ape” the relationship between eyes and pits, biometric measures