Biological Journal of the Linnean Society, 2011, , .

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Evolutionary losses of facial stripes in New World pitvipers

MATTHEW A. KWIATKOWSKI* and D. BRENT BURT
Department of Biology, Stephen F. Austin State University, P.O. Box 13003, Nacogdoches, TX 75962, USA
Received 9 March 2011; revised 30 June 2011; accepted for publication 30 June 2011
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Associations between the evolutionary loss or gain of colours and habitat can be informative about how environment drives colour evolution, and provide insight into their functions. Despite a wealth of studies on colours, the function of many colour patches are not well understood. This is especially the case for dark facial markings. Dark facial stripes are common in snakes, including many pitvipers, which possess facial stripes that extend from the eye towards the corner of the mouth. We investigated whether the evolutionary loss or gain of facial stripes in New World pitvipers is associated with activity period and habitat, hoping to shed light on their function. First, we examined whether evolutionary loss or gain of facial stripes is associated with nocturnal or diurnal activity. It has been suggested that facial stripes may protect the venom from ultraviolet radiation, which would be higher in diurnal species. Similarly, snakes in open habitats may experience higher ultraviolet radiation than those in closed habitats. Alternatively, we examined whether evolutionary loss or gain of facial stripes is associated with terrestrial or non-terrestrial habitat use. Studies on similar facial markings in other vertebrates indicated that they have a signalling function. If facial stripes in pitvipers have a signalling function, arboreal or saxicolous habitat use may limit signal effectiveness. Hence, we hypothesized that there may be an association between facial stripe evolution and habitat use. Using combined phylogenies that included almost every New World pitviper species, we tested correlations between facial stripes and ultraviolet exposure (activity period and habitat openness) and habitat use (terrestrial or non-terrestrial) using concentrated-changes tests and Pagels correlation tests. Our data did not support the hypothesis that ultraviolet exposure inuenced facial stripe evolution. Instead, the evolutionary loss of facial stripes was associated with non-terrestrial habitat use, such as arboreal and saxicolous habitats. In these habitats, we suggest the effectiveness of facial stripes as a signal would be limited, leading to their evolutionary loss. 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, , .

ADDITIONAL KEYWORDS: aposematic warning rattlesnakes signals ultraviolet radiation.

INTRODUCTION
Animal colours are inuenced by the environment in which they are displayed (Endler, 1990, 1992, 1993). Accordingly, habitats can inuence the evolutionary loss or gains of colours, resulting in phylogenetic patterns that can provide insight into how environment drives colour evolution (Wiens, 2000; Stoner, Bininda-Edmonds & Caro, 2003a; Caro 2009; Caro & Stankowich, 2010). Because colour often has a signalling function, a growing number of studies have found

*Corresponding author. E-mail: kwiatkowm@sfasu.edu

that habitat inuences the evolution of colour acting as social signals, especially mating signals, in birds, lizards, and mammals (Johnson & Lanyon, 2000; Badyaev & Hill, 2003; Ord & Stuart-Fox, 2005; Stuart-Fox, Moussalli & Whiting, 2007; Dolman & Stuart-Fox, 2010). Dark facial markings are found in many vertebrates and several studies suggest that they serve a signalling function, whether to potential predators as an aposematic warning, as suggested for some mammals (Newman, Buesching & Wolff, 2005), or to conspecics in social interactions (Ortolani, 1999; Stoner, Caro & Graham, 2003b; Caro & Stankowich, 2010). In birds, dark eye masks are inuenced by

2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, ,

M. A. KWIATKOWSKI and D. BRENT BURT function as warning or social/mating signals in other vertebrates (Stoner et al., 2003b; Newman et al., 2005; Tarof et al., 2005; Pederson et al., 2006; Kingma et al., 2008). These examples imply that facial stripes in pitvipers may have a signalling function, and that their evolution is associated with changes in habitat use. An association between habitat and the evolutionary loss or gain of facial stripes would provide important insight into the development of hypotheses regarding the selective forces acting on this colour pattern. Alternatively, the evolutionary loss or gain of facial stripes could be associated with exposure to ultraviolet radiation. Because facial stripes generally lie over the venom gland, Pough, Kwiecinski & Bemis (1978) argued that facial stripes in pitvipers help protect the venom from ultraviolet radiation. Despite the association between the venom gland and melanin pigment, the effects of ultraviolet radiation on venom have yet to be studied in situ or in vivo. Given that pitvipers are active during various times of the day, that they occupy a wide variety of habitats, and that some species have broad geographic distributions, it is likely that exposure to ultraviolet radiation varies considerably among and within species. Because melanin production can be costly (Jawor & Breitwisch, 2003), evolutionary losses of facial stripes may occur when exposure to ultraviolet radiation is limited in closed habitats, or evolutionary gains of facial stripes may only occur under conditions of high ultraviolet in open habitats. We examined the evolution of facial stripes to gain insight into the possible selective forces acting on them. First, we examined whether ultraviolet exposure, as determined by time of activity and habitat openness, inuences the evolution of facial stripes. We predicted that the evolutionary loss of facial stripes would be associated with nocturnal activity or closed (low-ultraviolet) habitats, whereas evolutionary gains would be associated with diurnal activity or open (high-ultraviolet) habitats. Second, we examined the inuence of habitat use on facial stripe evolution. We suggest that an association could indicate a fruitful avenue for future testing whether facial stripes act as signals. If facial stripes are used as signals that are effective only in terrestrial habitats, then we would expect their evolutionary loss more often in lineages using non-terrestrial habitats. For example, arboreal and precipitous rocky environments could preclude any function of facial stripes as a social signal, because social interactions among snakes, such as male combat or mate assessment, would be limited in such environments. Similarly, if facial stripes function as a warning signal, their utility may be reduced in arboreal and precipitous

malemale competition, by female mate choice, or by both of these drivers (Tarof, Dunn & Whittingham, 2005; Leichty & Grier, 2006; Pederson, Dunn & Whittingham, 2006; Pogny & Szkely, 2007; Kingma et al., 2008). Because these dark markings are often located near or around the eyes, an alternative explanation is that they function in ecophysiological contexts, such as camouaging the eye for protection, reducing glare in bright light, or for targeting prey items (see the review by Ficken, Matthiae & Horwich, 1971; Ortolani, 1999). Despite growing interest in the function of dark facial markings, relatively few studies have investigated how their evolution might be associated with habitat. When their evolution is examined in a phylogenetic context, dark facial colours in mammals are associated with different habitats, depending on the particular group, and appear to have signalling functions (Ortolani, 1999; Caro & Stankowich, 2010). Dark facial markings in various snakes occur as facial stripes, which typically originate near the eye and extend posteriorly and laterally to approximately the corner of the mouth. Facial stripes are widespread in the pitvipers (Viperidae: Crotalinae), but unlike other vertebrates, they are little studied and no denitive function has been described. Generally, facial stripes in pitvipers vary from a stripe that is somewhat darker than the background colour to a black band bordered by narrower white stripes. In arboreal snakes with dark lines anterior to the eyes, some have suggested the eye stripes may function in prey sighting (Ficken et al., 1971). However, facial stripes in almost all pitvipers are found posterior to the eyes, precluding their function as a targeting mechanism. Still, the observation by Ficken et al. (1971) that facial stripes could be associated with a particular habitat provides a potential starting point for investigating whether habitat inuences the evolution of facial stripes, which could ultimately provide insight into their function. Pitvipers occupy a diversity of habitats and although most are terrestrial, it is not uncommon for species to occupy arboreal or saxicolous habitats. In some reptiles, similar differences in habitat use are associated with evolutionary gains or losses of colour patterns and other types of displays. In phrynosomatid lizards, Wiens (1999) found that the few gains and many losses in male signals were associated with terrestrial rather than arboreal or saxicolous habitats. In Asian pitvipers, some arboreal species have evolved red colour patterns, which may result from Mllerian mimicry (Sanders, Malhotra & Thorpe, 2006). Furthermore, pitviper facial stripes t the criteria necessary for being conspicuous (Endler, 1990, 1992) and are similar to dark facial markings that

2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, ,

FACIAL STRIPES IN NEW WORLD PITVIPERS rocky environments, where a predator may view a snake from much different angles compared with a terrestrial snake.

MATERIAL AND METHODS FACIAL STRIPE SCORES

Facial stripe data were collected by scoring photographs from two sources: Campbell & Lamar (1989, 2004). Campbell & Lamar (2004) was especially useful in that it contained at least one photograph, often more, for almost every species found in the phylogenies used in this study (Castoe & Parkinson, 2006; Fenwick et al., 2009; see below). In some cases, the same photographs were used in both sources, so care was taken to ensure that a photograph was not scored multiple times. As facial stripes were scored by one of the authors (M.A.K.), we tested whether the scores were objective by comparison with a second observer. The second observer was an undergraduate volunteer who received instructions regarding the criteria for assigning scores, but had no prior knowledge of the hypotheses of the study. Although criteria were set for determining the facial stripe score, it is likely that some subjectivity existed between observers. Hence, to determine consistency across observers, the correlation between scores from the two observers was investigated, and separate phylogenetic analyses were conducted using scores from each observer. These analyses were conducted on a smaller phylogeny (Castoe & Parkinson, 2006; see the Phylogenies section below) using the same methods as the combined, more comprehensive, phylogenies used for the full analyses, yet the technique was the same. Photographs were scored on a scale of 15 based on the apparent conspicuousness of the facial stripe to a human observer, with a score of 1 representing no facial stripe and a score of 5 being the most conspicuous. If more than one photograph was available for a species, each photograph was scored and the mean score was calculated and used in subsequent analyses. Because more than one observer was used for collecting data, certain criteria were dened for each score to maintain as much consistency between observers as possible. These criteria were based on the contrast between the facial stripe and the background head colour, because contrast is a fundamental means of increasing the conspicuousness of a colour pattern, regardless of the visual system of the receiver (Endler, 1990, 1992). Facial stripe contrast can be a function of two mechanisms: how dark the facial stripe is relative to the scales found on the lateral sides of the head (i.e. the background head colour) or whether the facial stripe is bordered by

scales much lighter in colour (as described by Endler, 1992). Facial stripe contrast in most pitvipers is the result of the rst mechanism, although some striking examples can be found resulting from the second mechanism (e.g. Agkistrodon bilineatus and Agkistrodon taylori). Facial stripe scores were dened as: 1, no facial stripe visible; 2, faint facial stripe visible, but little contrast with background head colour; 3, moderate contrast between stripe and background head colour; 4, facial stripe contrasting with background head colour, stripe may be bordered by lighter scales; and 5, facial stripe distinctly contrasting with background head colour or facial stripe bordered by scales much lighter in colour than the facial stripe. Generally, darker facial stripes were more conspicuous than lighter stripes, although this was not necessarily the case for a few species where dark markings other than the stripe were found on the head. Because the phylogenetic analyses used in this study required binary data, scores of 1 and 2 were considered 0, meaning an absence of a facial stripe, whereas scores of 35 were considered a 1, indicating the presence of a facial stripe. Although this does not divide the original scores evenly, it seems to best reect the appearance of the facial stripe, given that the observers noticed any stripe with a score over 2 seemed readily visible.

ULTRAVIOLET

EXPOSURE AND TIME OF ACTIVITY

The level of ultraviolet exposure can be inuenced by time of activity. Strictly nocturnal snakes will have no exposure, whereas strictly diurnal snakes may encounter considerable ultraviolet radiation. Species were scored as nocturnal (0) or diurnal (1) using primarily the same sources used to score facial stripe (Klauber, 1972; Campbell & Lamar, 1989, 2004). Many species, however, cannot be classied as either nocturnal or diurnal, as it is not uncommon for pitvipers to be active, or at least out, during the day and at night. Hence, many species were scored as both nocturnal and diurnal (see analyses below). For most species in the large genus Bothrops, only anecdotal observations are available for activity time. Campbell & Lamar (2004) generalized activity for this genus based on elevation, with species found at lower elevations being nocturnal and those at higher elevations being diurnal. We followed this generalization for most Bothrops species, given the limited availability of denitive data. Accordingly, species found primarily below 1000 m a.s.l. were scored as nocturnal, whereas species found primarily above 1000 m a.s.l. were scored as diurnal, unless specic observations suggested otherwise. Because so many species were scored as both nocturnal and diurnal, both ACCTRAN and DELTRAN

2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, ,

M. A. KWIATKOWSKI and D. BRENT BURT terrestrial and non-terrestrial habitats. These species present problems for use in Pagels correlation test (Pagel, 1994, see below) because it cannot include taxa with polymorphic character states. In these cases (e.g. Crotalus molossus, Crotalus ruber, and Bothriechis aurifer), each polymorphic species was scored as being terrestrial in one analysis and non-terrestrial in a second analysis. In other cases, species were classied based on their primary habitat. For example, Crotalus horridus, which is known to climb trees, was classied as terrestrial because most of its habitat use is terrestrial, especially in adulthood (Rudolph et al., 2004). In one instance, specic reference was made to Ophryacus undulatus being more arboreal than other members of the genus (Campbell & Lamar, 2004). In this case, the species was classied as arboreal.

reconstructions produced numerous equivocal branches. Hence, separate analyses were run after manually assigning these branches as either nocturnal or diurnal.

ULTRAVIOLET

EXPOSURE AND HABITAT OPENNESS

Activity time (diurnal or nocturnal) is not necessarily a good indicator of ultraviolet exposure. Diurnal snakes are not necessarily exposed to high levels of ultraviolet radiation if they are found in habitats where solar exposure is limited. Hence, a better measure of ultraviolet exposure may be habitat openness. Solar radiation intensity, including ultraviolet wavelengths, increases with decreasing latitude. Accordingly, pitvipers at lower latitudes would presumably experience higher levels of ultraviolet radiation. However, effects of latitude are confounded by cloud cover and vegetation cover, which will be highest at the equator. Increasing cloud and vegetation cover will reduce the absolute levels of both direct and diffuse ultraviolet irradiance (Endler, 1993; Parisi et al., 2000). Hence, pitvipers in open habitats with little cloud cover and vegetation, such as deserts, will generally experience higher levels of ultraviolet irradiance compared with forests. For the phylogenetic analyses, we scored pitvipers found in relatively open habitats, such as deserts, with a 1 for high ultraviolet exposure, and those found in forested habitats as a 0 for low ultraviolet exposure. Ultraviolet exposure can be difficult to score for certain habitats. For example, some forests may be open enough such that ultraviolet exposure could be high, even though it is a forested habitat (e.g. pineoak forest). To account for this uncertainty, we conducted two different analyses. For the rst analysis, taxa in habitats with uncertain ultraviolet exposure were scored as occupying low ultraviolet exposure habitats. For the second analysis, the same taxa were scored as occupying high ultraviolet exposure habitats.

PHYLOGENIES
Although various phylogenies exist for the pitvipers, Castoe & Parkinson (2006) is the most extensive. They present both a strict consensus maximum parsimony tree (of 12 equally parsimonious trees) and a Bayesian consensus tree based on the analysis of 2306 bp of mitochondrial DNA (mtDNA). The maximum-parsimony tree of Castoe & Parkinson (2006) does not show neartic pitvipers as monophyletic, contrary to most other phylogenies (e.g. Gutberlet & Harvey, 2002; Parkinson, Campbell & Chippendale, 2002). However, this gives our analysis the benet of testing two phylogenies: one in which the neartic pitvipers are monophyletic (the Bayesian consensus tree), consistent with other published phylogenies, and a phylogeny in which the neartic pitvipers are not monophyletic. Analyses were conducted using phylogenies from Castoe & Parkinson (2006), but the clade containing Bothrops, Bothriopsis, and Bothrocophias was replaced with a much more extensive phylogeny of those three genera from Fenwick et al. (2009), increasing the number of species for that clade from 14 to 39. With the combined phylogenies, the analyses included almost all New World pitviper species. The polytomy of Crotalus adamanteus, Crotalus mitchelli, and Crotalus tigris from the original parsimony tree of Castoe & Parkinson (2006) was modied in our analyses such that C. mitchelli and C. tigris were made sister taxa, and C. adamanteus was made sister to the C. mitchelli/C. tigris clade. We resolved the polytomy in this manner for two reasons: rst, it follows other phylogenies of the rattlesnakes (e.g. Murphy et al., 2002); and second, C. mitchelli and C. tigris are sympatric in the south-western USA, whereas C. adamanteus is limited to south-eastern USA. The polytomy of the Bothriechis, Ophryacus/

HABITAT

USE

Species were assigned one of three forms of habitat use: terrestrial, arboreal, or saxicolous. Habitat used by each species was determined from the natural history descriptions found in Campbell & Lamar (1989, 2004). Species were classied as terrestrial if they are primarily found on the ground or nonterrestrial if they were arboreal or saxicolous (see further details below). Species were only classied as non-terrestrial if a reference (or personal experience, M.A.K.) indicated that most of their time was spent in that habitat. Some species are known to occupy

2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, ,

FACIAL STRIPES IN NEW WORLD PITVIPERS Lachesis, and Agkistrodon/Sistrurus/Crotalus clades in the original Bayesian tree was resolved as indicated in Castoe et al. (2009): the Bothriechis clade became basal to a clade comprised of the Ophryacus/ Lachesis and Agkistrodon/Sistrurus/Crotalus sister clades.

CHARACTER

EVOLUTION ANALYSES

Evolutionary changes in facial stripe score, habitat, and ultraviolet environment were reconstructed in MACCLADE 4.08 (Maddison & Maddison, 2005) using the pitviper maximum-parsimony and Bayesian consensus phylogenies of Castoe & Parkinson (2006), modied to include the species from Fenwick et al. (2009). ACCTRAN and DELTRAN algorithms were used to resolve equivocal ancestral character state assignments: ACCTRAN accelerates transitions, whereas DELTRAN delays transitions. We conducted all analyses using both ACCTRAN and DELTRAN, as a recent study suggested that neither minimizes parallel gain of characters (Agnarsson & Miller, 2008). The presence of facial stripes was reconstructed as the basal state in each phylogeny, with 15 or 17 losses and one or three gains. Such a small number of gains limits our ability to test the hypothesis that facial stripes evolve in terrestrial habitats. Accordingly, the loss of facial stripes became the evolutionary change of interest (Johnson & Lanyon, 2000). Therefore, we tested whether losses of facial stripes are linked to nocturnal activity, low-ultraviolet environments, or non-terrestrial habitats. We used concentratedchanges tests (Maddison, 1990) in MACCLADE 4.08 (Maddison & Maddison, 2005) to test our hypotheses. For each phylogeny (parsimony and Bayesian), we examined the probability of getting the reconstructed number of losses of eye stripes (dependent character) in branches representing nocturnal activity, lowultraviolet environments, or non-terrestrial habitats (independent character), assuming a null model in which each character evolved independently of one another. Our a priori hypotheses are supported when losses of facial stripes are over-represented in lineages representing nocturnal activity, low-ultraviolet environments, or non-terrestrial habitats. The concentrated-changes test requires the use of binary characters and we dened our independent character as non-terrestrial for precipitous saxicolous and arboreal habitats, as distinct from more level terrestrial habitats, because both arboreal and precipitous saxicolous environments could limit the visibility and mobility of snakes, precluding the use of facial stripes as a potential signal. Five of the 99 species used in these analyses are known to vary in their use of terrestrial and non-terrestrial habitats, and they were coded as polymorphic (i.e. terrestrial

and non-terrestrial). We used 10 000 simulations for each concentrated-changes test. Finally, we used Pagels correlation test (Pagel, 1994) using the CORREL package in MESQUITE 2.74 (build 550; Maddison & Maddison, 2010) to investigate whether the evolution of our characters of interest show correlated patterns of evolution. This test differs from the concentrated-changes test in that it considers branch lengths and uses likelihood methods to test for patterns of correlated evolution between two binary characters. Branch lengths were only available for the Bayesian consensus tree of Castoe & Parkinson (2006), so only results for this smaller tree are reported. Like the concentratedchanges tests above, analyses were performed for data from both observers. Each test used 1000 simulations in its calculations. Unlike the concentratedchanges test, the Pagels test does not work for taxa with polymorphic character states. Four taxa were polymorphic for either ultraviolet environment or habitat use; therefore, two Pagel analyses were performed for both ultraviolet and signalling habitat hypotheses. The rst analysis of the ultraviolet hypothesis scored all polymorphic species as occupying closed habitat (low ultraviolet exposure), whereas the second analysis scored them as occupying open habitat (high ultraviolet exposure). The rst analysis of the signalling hypothesis scored all polymorphic species as terrestrial, whereas the second analysis scored them as non-terrestrial. Pagels test was not feasible for testing correlations of facial stripe loss with nocturnal activity because of the large number of taxa with polymorphic character state assignments.

RESULTS
Facial scores were highly correlated between the two observers for the rst set of analyses using only Castoe & Parkinsons (2006) phylogenies (Pearson correlation coefficient = 0.86), suggesting that facial stripe conspicuousness can be consistently quantied, at least by human observers. The second observer was not available for scoring stripes from the phylogeny of Fenwick et al. (2009). However, results were highly consistent among the two observers, with only two tests out of 16 differing in signicance (test results can be provided by the authors, upon request). Hence, bias did not appear to be a problem and, if anything, results based on the authors (M.A.K.) scoring were more conservative. Reconstructions showed that presence of facial stripes is the ancestral condition for New World pitvipers (Figs 1, 2) for both the maximum-parsimony and Bayesian trees. The ancestral condition for daily activity (diurnal versus nocturnal) was equivocal for both trees. Closed habitat (low ultraviolet exposure) is

2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, ,

M. A. KWIATKOWSKI and D. BRENT BURT

Ophryacus undulatus Ophryacus melanurus Lachesis stenophrys Lachesis muta Agkistrodon contortrix Agkistrodon piscivorus Agkistrodon bilineatus Agkistrodon taylori Bothriechis schlegelii Bothriechis supraciliaris Bothriechis nigroviridis Bothriechis lateralis Bothriechis bicolor Bothriechis thalassinus Bothriechis marchi Bothriechis aurifer Bothriechis rowleyi Atropoides occiduus Atropoides olmec Atropoides mexicanus Atropoides nummifer Atropoides picadoi Cerrophidion petlalcalensis Cerrophidion godmani Porthidium ophryomegas Porthidium dunni Porthidium nasutum Porthidium porrasi Porthidium arcose Bothrocophias hyoprora Bothrocophias myersi Bothrocophias campbelli Bothrops venezuelensis Bothrops pictus Bothrops andianus Bothrops ammodytoides Bothrops cotiara Bothrops fonsecai Bothrops alternatus Bothrops mattogrossensis Bothrops itapetiningae Bothrops jonathani Bothrops insularis Bothrops alacatraz Bothrops jararaca Bothrops sanctaecrucis Bothrops erythromelas Bothrops neuwiedi Bothrops diporus Bothrops pauloensis Bothriopsis chloromelas Bothriopsis taeniata Bothriopsis pulchra Bothriopsis bilineata Bothrops barnetti Bothrops brazili Bothrops jararacussu Bothrops osbornei Bothrops punctatus Bothrops caribbaeus Bothrops lanceolatus Bothrops asper Bothrops marajoensis Bothrops moojeni Bothrops leucurus Bothrops atrox Bothrops isabelae Sistrurus catenatus Sistrurus miliarius Crotalus polystictus Crotalus cerastes Crotalus ravus Crotalus pusillus Crotalus triseriatus Crotalus lepidus Crotalus aquilus Crotalus triseriatus (LG) Crotalus molossus Crotalus basiliscus Crotalus enyo Crotalus unicolor Crotalus durissus Crotalus vegrandis Crotalus exsul Crotalus ruber Crotalus catalinensis Crotalus tortugensis Crotalus atrox Crotalus horridus Crotalus pricei Crotalus intermedius Crotalus transversus Crotalus willardi Crotalus adamanteus Crotalus tigris Crotalus mitchelli Crotalus scutulatus Crotalus oreganus

Bothrocophias microphthalmus

No Facial Stripe Facial Stripe Present Equivocal Polymorphic taxa

Non-terrestrial Terrestrial Equivocal Polymorphic taxa

Figure 1. Strict consensus maximum-parsimony tree from Castoe & Parkinson (2006), with the clade representing Bothrops, Bothriopsis, and Bothrocophias species replaced by the phylogeny from Fenwick et al. (2009), showing the most parsimonious reconstructions of the evolution of facial stripes on the left, and the parsimony reconstructions of the evolution of non-terrestrial habitat use (i.e. arboreal or saxicolous) on the right.

the ancestral condition using the Bayesian tree, but the ancestral condition was equivocal with the maximum-parsimony tree. Use of terrestrial habitats is the ancestral condition using both the maximumparsimony and Bayesian trees. Facial stripes were lost 15 times for both the maximum-parsimony and Bayesian consensus tree ACCTRAN reconstructions, and 17 times using DELTRAN reconstructions (Figs 1, 2; Table 1). Despite considerable differences between the maximumparsimony and Bayesian consensus trees, results from the concentrated-changes tests were very similar (Table 1). There was no trend for nocturnal snakes to lose facial stripes, nor was there any effect of low-ultraviolet habitats. Evolutionary losses of facial stripes were not associated with nocturnal

activity or low ultraviolet exposure in any of the tests. Rather, the evolutionary loss of facial stripes was associated with non-terrestrial habitat use with the DELTRAN reconstructions for both the maximum-parsimony and Bayesian trees. Nonterrestrial habitat use occurs in the typically arboreal genera Bothriechis and Bothriopsis, as well as in some saxicolous rattlesnake species (Figs 1, 2). Pagels correlation tests using the smaller phylogeny revealed similar results to the concentratedchanges tests. Both tests for an association between the evolution of facial stripes and terrestrial versus non-terrestrial habitat use were highly signicant (P < 0.001, for both tests where polymorphic species were rst scored as non-terrestrial and then scored as terrestrial for the second analysis). Neither test for an

2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, ,

FACIAL STRIPES IN NEW WORLD PITVIPERS

Atropoides occiduus Atropoides olmec Atropoides mexicanus Atropoides nummifer Atropoides picadoi Cerrophidion petlalcalensis Cerrophidion godmani Porthidium ophryomegas Porthidium dunni Porthidium nasutum Porthidium porrasi Porthidium arcose Bothrocophias hyoprora Bothrocophias myersi Bothrocophias campbelli Bothrops venezuelensis Bothrops pictus Bothrops andianus Bothrops ammodytoides Bothrops cotiara Bothrops fonsecai Bothrops alternatus Bothrops mattogrossensis Bothrops itapetiningae Bothrops jonathani Bothrops insularis Bothrops alacatraz Bothrops jararaca Bothrops sanctaecrucis Bothrops erythromelas Bothrops neuwiedi Bothrops diporus Bothrops pauloensis Bothriopsis chloromelas Bothriopsis taeniata Bothriopsis pulchra Bothriopsis bilineata Bothrops barnetti Bothrops brazili Bothrops jararacussu Bothrops osbornei Bothrops punctatus Bothrops caribbaeus Bothrops lanceolatus Bothrops asper Bothrops marajoensis Bothrops moojeni Bothrops leucurus Bothrops atrox Bothrops isabelae Bothriechis schlegelii Bothriechis supraciliaris Bothriechis nigroviridis Bothriechis lateralis Bothriechis thalassinus Bothriechis marchi Bothriechis bicolor Bothriechis aurifer Bothriechis rowleyi Ophryacus undulatus Ophryacus melanurus Lachesis stenophrys Lachesis muta Agkistrodon contortrix Agkistrodon piscivorus Agkistrodon bilineatus Agkistrodon taylori Sistrurus catenatus Sistrurus miliarius Crotalus polystictus Crotalus cerastes Crotalus ravus Crotalus pusillus Crotalus triseriatus Crotalus lepidus Crotalus aquilus Crotalus triseriatus (LG) Crotalus pricei Crotalus intermedius Crotalus transversus Crotalus horridus Crotalus willardi Crotalus adamanteus Crotalus tigris Crotalus mitchelli Crotalus scutulatus Crotalus oreganus Crotalus atrox Crotalus tortugensis Crotalus catalinensis Crotalus exsul Crotalus ruber Crotalus enyo Crotalus molossus Crotalus basiliscus Crotalus unicolor Crotalus durissus Crotalus vegrandis

Bothrocophias microphthalmus

No Facial Stripe Facial Stripe Present Equivocal Polymorphic taxa

Non-terrestrial Terrestrial Equivocal Polymorphic taxa

Figure 2. Bayesian consensus trees from Castoe & Parkinson (2006), with the clade representing Bothrops, Bothriopsis, and Bothrocophias species replaced by the phylogeny from Fenwick et al. (2009), showing the most parsimonious reconstructions of the evolution of facial stripes on the left, and the parsimony reconstructions of the evolution of non-terrestrial habitat use (i.e. arboreal or saxicolous) on the right.

association between the evolution of facial stripes and habitat type based on ultraviolet exposure was signicant (P = 0.208, with polymorphic species scored as occupying low-ultraviolet habitats and P = 0.129 with polymorphic species scored as occupying highultraviolet habitats).

in colour patterns to be associated with habitat type. However, this study is the rst to demonstrate this in snakes.

FACIAL

STRIPES AND ULTRAVIOLET EXPOSURE

DISCUSSION
Our results indicate that evolutionary losses of facial stripes in New World pitvipers are associated with non-terrestrial habitat use rather than low ultraviolet exposure. These results join a growing number of studies that have found evolutionary gains or losses

There was no association between loss of facial stripes and nocturnal activity. The lack of an association between facial stripe evolution and time of activity is not surprising. Many species exhibit both nocturnal and diurnal activity, and even diurnal snakes will not necessarily have high exposure to ultraviolet if they can avoid exposure during the day. Although we found no effects of ultraviolet exposure, some important caveats, both for and against, should be mentioned.

2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, ,

M. A. KWIATKOWSKI and D. BRENT BURT

Table 1. Results of concentrated-changes tests using ACCTRAN and DELTRAN reconstructions for both the maximumparsimony and Bayesian consensus trees from Castoe & Parkinson (2006), but with the clade representing Bothrops, Bothriopsis, and Bothrocophias species replaced by the more extensive phylogeny from Fenwick et al. (2009) Maximum parsimony ACCTRAN (3G/15L) 8L P = 0.230 9L P = 0.240 11L P = 0.120 10L P = 0.07 6L P = 0.104 DELTRAN (1G/17L) 7L P = 0.223 12L P = 0.241 13L P = 0.0724 11L P = 0.10 8L P = 0.0222 Bayesian ACCTRAN (3G/15L) 10L P = 0.240 10L P = 0.240 11L P = 0.128 10L P = 0.08 6L P = 0.0985 DELTRAN (1G/17L) 7L P = 0.225 12L P = 0.241 13L P = 0.0813 11L P = 0.10 8L P = 0.0236

Test habitat Nocturnal (Equivocal branches assigned a nocturnal state) Nocturnal (Equivocal branches assigned a diurnal state) Low ultraviolet (Taxa in habitats with unclear UV exposure scored as low UV) Low ultraviolet (Taxa in habitats with unclear UV exposure scored as high UV) Non-terrestrial

Tests highlighted in bold show signicant associations between losses (L) in facial stripes and the associated test habitat. Two tests for both nocturnal activity pattern and low UV exposure were done due to uncertainty in reconstructions and degree of UV exposure in certain habitats.

First, the habitat tests we used required categorical data, which oversimplies ultraviolet conditions into high or low: ultraviolet conditions experienced by species in such a broadly distributed clade are clearly more complex. Quantifying ultraviolet in tropical regions for each species would be especially difficult because of the undoubtedly intricate ultraviolet conditions many experience. For example, in tropical deciduous forests, there is little continuous canopy during the dry season, whereas the canopy of tropical evergreen forest is relatively continuous year round (Endler, 1992). Accordingly, pitvipers in tropical evergreen forests experience widespread and constant shade, suggesting that ultraviolet radiation would have little impact on venom, whereas snakes in tropical dry forests may experience higher levels of ultraviolet radiation during the dry season. Although we generalized ultraviolet conditions by habitat, the actual conditions will be a function of behaviour, which is little known in many species. Whereas some of these oversimplications may bias our tests against the ultraviolet hypothesis, other generalizations may have biased our results in favour of an ultraviolet effect. For example, most of the rattlesnakes were categorized as experiencing high ultraviolet exposure. Indeed, the arid habitats where many rattlesnakes are found are relatively open and ultraviolet radiation is high. However, rattlesnakes are generally crepuscular or nocturnal (Klauber, 1972); hence, their activity peaks during the hours

when ultraviolet radiation is lowest. Thus, many pitvipers found in habitats with some of the highest ultraviolet radiation do not actually experience high exposure, supporting the argument that ultraviolet radiation has a limited role in the evolution of facial stripes. Despite our results indicating that facial stripes per se are not associated with ultraviolet protection, our ndings are not necessarily completely contradictory to Pough et al.s (1978) morphological study. Although Pough et al. (1978) suggested that facial stripes are likely contribute to ultraviolet protection, especially along the anterolateral aspects of the venom gland, the extent of melanin deposits in deeper layers may compensate for a weak facial stripe. Whether these melanin deposits actually protect the venom has yet to be determined: they may simply be the result of the same developmental pathways that regulate the facial stripe and, therefore, may not be associated with ultraviolet protection. Furthermore, whether ultraviolet actually damages venom in situ or in vivo is not well understood, although in vitro exposure to ultraviolet radiation can degrade the venom (Macht, 1935; Tejasen & Ottolenghi, 1970).

FACIAL

STRIPES AND HABITAT USE

We found evidence of an association between the evolutionary loss of facial stripes and non-terrestrial

2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, ,

FACIAL STRIPES IN NEW WORLD PITVIPERS habitat use. Effects of habitat on colour patterns have been found in various vertebrate clades (e.g. Ortolani, 1999; Stuart-Fox, Moussalli & Whiting, 2007; Caro, 2009), although, to the best of our knowledge, this is the rst phylogenetic analysis of such a relationship in snakes. Glare effects are probably limited in heavily forested habitats, but could be large in open habitats such as deserts. Given the primarily nocturnal nature of most desert species, glare is not likely to be a debilitating problem. Additionally, the position of pitviper facial stripes posterior to the eyes in almost all species in this study would preclude inuences of prey targeting (e.g. Ficken et al., 1971) and glare reduction (e.g. Ortolani, 1999). Recent studies have found an association between habitat and the evolutionary loss or gain of colour patterns that function in signalling. In blackbirds, evolutionary gains of colour signals are associated with marsh habitats (Johnson & Lanyon, 2000). In phrynosomatid lizards, the evolutionary loss of display signals is associated with the use terrestrial habitats as opposed to rocky or arboreal ones (Wiens, 1999). Interestingly, Wiens (1999) results for phrynosomatid lizards are the opposite of our results for snakes, where we found that facial stripes are lost in arboreal and saxicolous habitats rather than in terrestrial ones. In snakes, it is speculative to suggest a potential signalling function of facial stripes and why they might be lost in non-terrestrial habitats. Facial stripe loss in non-terrestrial species may result from selective pressures to be more cryptic. Signals typically increase conspicuousness, and if facial stripes have a signalling function, they may draw attention to the head. Indeed, facial stripes of some species in this study would be quite conspicuous, regardless of the observer, given the large contrast between light and dark components (Endler, 1990, 1992). Considering evidence for similar facial markings found in other vertebrates, two clear contexts for signalling arise: warning and social signalling.

simply making the head more conspicuous, similar to the zigzag body pattern found in some vipers (Wster et al., 2004; Niskanen & Mappes, 2005). In Asian pitvipers, some arboreal species have evolved conspicuous red colour patterns (Sanders et al., 2006). In these species, a conspicuous red stripe, sometimes bordered by a white stripe, extends down the lateral side of the body, and a red facial stripe may also be found in some of these species that matches the body stripe. Sanders et al. (2006) argued that this stripe is the result of Mllerian mimicry, implying a warning signal function. The evolution of red stripes in arboreal Asian pitvipers seems contradictory to our ndings of facial stripe loss in arboreal New World pitvipers. Further ecological and evolutionary comparisons of these clades could prove productive towards understanding the function of these colour patterns, and the inuence of habitat on their evolution. If facial stripes in New World pitvipers function as a warning signal, our results imply that nonterrestrial species experience different predator threats compared with terrestrial species. Perhaps terrestrial pitvipers encounter more predation attempts, or face different predators, compared with those in non-terrestrial habitats. Mammals and raptors are likely to be the most frequent predators of pitvipers (Greene, 1992, 1997), but little has been documented on predation differences among habitats. Alternatively, the loss of facial stripes in nonterrestrial species may result from selective pressures to be more cryptic.

SOCIAL

SIGNAL

WARNING

SIGNAL

Facial stripes of pitvipers are similar to the facial markings found in many mammals, a group in which the evolution of dark facial markings of some species is associated with habitat (Ortolani, 1999). In midguild mammalian carnivores, facial masks may function as aposematic signals to potential predators, especially larger mammals (Newman et al., 2005). Pitvipers also face threats from larger mammalian predators (among others; see Greene, 1992, 1997), and they certainly possess the ability to harm a larger predator. Facial stripes may function as a visual warning signal by accentuating head dimensions or

In many vertebrates, dark facial markings function in social signalling. In mammals, studies using phylogenetic techniques similar to ours indicate that dark facial colours may be used in conspecic communication (Stoner et al., 2003b; Caro & Stankowich, 2010). In birds, dark eye masks, especially their size, are inuenced by sexual selection (Tarof et al., 2005; Leichty & Grier, 2006; Pederson et al., 2006; Pogny & Szkely, 2007; Kingma et al., 2008). If facial stripes are a sexually selected signal, their loss in arboreal and saxicolous species suggests that signalling is not informative in those habitats. Perhaps signal efficacy is low in non-terrestrial species because the snake is generally less visible or likely to be observed from above or below, thereby limiting the view of the facial stripes. Alternatively, as described above, the loss of facial stripes in non-terrestrial species may result from selective pressures to be more cryptic. Ultimately, concrete evidence for a signalling function of facial stripes will require behavioural and ecological studies.

2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, ,

10

M. A. KWIATKOWSKI and D. BRENT BURT

ACKNOWLEDGEMENTS
We thank E. Fucik for help with data collection, T. Castoe for providing branch lengths for a phylogeny, and B. Sullivan for critically reading the article.

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