Facultative thermogenesis during brooding is not the norm among pythons

Abstract

Facultative thermogenesis is often attributed to pythons in general despite limited comparative data available for the family. While all species within Pythonidae brood their eggs, only two species are known to produce heat to enhance embryonic thermal regulation. By contrast, a few python species have been reported to have insignificant thermogenic capabilities. To provide insight into potential phylogenetic, morphological, and ecological factors influencing thermogenic capability among pythons, we measured metabolic rates and clutch-environment temperature differentials at two environmental temperatures--python preferred brooding temperature (31.5 °C) and a sub-optimal temperature (25.5 °C)--in six species of pythons, including members of two major phylogenetic branches currently devoid of data on the subject. We found no evidence of facultative thermogenesis in five species: Aspidites melanocephalus, A. ramsayi, Morelia viridis, M. spilota cheynei, and Python regius. However, we found that Bothrochilus boa had a thermal metabolic sensitivity indicative of facultative thermogenesis (i.e., a higher metabolic rate at the lower temperature). However, its metabolic rate was quite low and technical challenges prevented us from measuring temperature differential to make conclusions about facultative endothermy in this species. Regardless, our data combined with existing literature demonstrate that facultative thermogenesis is not as widespread among pythons as previously thought.

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Web End = Facultative thermogenesis during brooding is not the norm among pythons

Jake Brashears1,2 Dale F. DeNardo1

Abstract Facultative thermogenesis is often attributed to pythons in general despite limited comparative data available for the family. While all species within Pythonidae brood their eggs, only two species are known to produce heat to enhance embryonic thermal regulation. By contrast, a few python species have been reported to have insignicant thermogenic capabilities. To provide insight into potential phylogenetic, morphological, and ecological factors inuencing thermogenic capability among pythons, we measured metabolic rates and clutch-environment temperature differentials at two environmental temperaturespython preferred brooding temperature (31.5 C) and a sub-optimal temperature (25.5 C)in six species of pythons, including members of two major phylogenetic branches currently devoid of data on the subject. We found no evidence of facultative thermogenesis in ve species: Aspidites melanocephalus, A. ramsayi, Morelia viridis, M. spilota cheynei, and Python regius. However, we found that Bothrochilus boa had a thermal metabolic sensitivity indicative of facultative thermogenesis (i.e., a higher metabolic rate at the lower temperature). However, its metabolic rate was quite low and technical challenges prevented us from measuring temperature differential to make conclusions about facultative endothermy in this species. Regardless,

Received: 8 February 2015 / Revised: 2 May 2015 / Accepted: 12 June 2015 / Published online: 27 June 2015 Springer-Verlag Berlin Heidelberg 2015

our data combined with existing literature demonstrate that facultative thermogenesis is not as widespread among pythons as previously thought.

Keywords Evolution of endothermy Metabolic rate

Parental care Reptile Snake

Introduction

Although it often requires a substantial expenditure of resources on the part of the parent, parental care is widespread because it confers numerous tness benets to offspring (e.g., increased survival and growth) (Clutton-Brock 1991). A common benet of parental care is thermoregulation of the developing offspring (Shine and Harlow 1996;Ashmore and Janzen 2003). In fact, thermoregulation of the developmental environment has been proposed as an initial driving factor in the evolution of endothermy (Farmer 2000).

Pythons (Squamata: Pythonidae) are rare among reptiles in providing ubiquitous parental care that provides thermal, hydric, and predator defense benets (Shine 2004;Stahlschmidt and DeNardo 2010). Pythons are ectothermic throughout most of life, but during brooding the females of some species are known to increase their metabolic rate and exhibit muscular twitching, termed facultative thermogenesis. While widely accepted as a trait of pythons in general, of the 44 extant python species (Barker et al. 2015), facultative thermogenesis has been conrmed in only two species: the Burmese python (Python molurus) (Vinegar et al. 1970), which has since been broken into two species (P. molurus and P. bivitattus, Jacobs et al. 2009) and two sub-species of the carpet python (Morelia spilota spilota) (Slip and Shine 1988); (M. s. imbricata) (Pearson et al. 2003). In

* Jake Brashears [email protected]

Dale F. DeNardo [email protected]

1 School of Life Sciences, Arizona State University, PO Box 874501, Tempe, AZ 85281-4501, USA

2 Present Address: Department of Life Sciences, San Diego City College, 1313 Park Boulevard, San Diego, CA 92101-4787, USA

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these species, facultative thermogenesis provides substantial heat to the clutch, supplementing the insulatory benets associated with coiling around the eggs (Stahlschmidt et al. 2008). Female heat production is inversely proportional to environmental temperature (Tenv), with metabolic rate and muscular twitching increasing as temperature decreases below optimal developmental temperature (approximately 31.5 C for most species) (Brashears and DeNardo 2013). In P. molurus, maximum heat production occurs when Tenv

approaches 24 C (Van Mierop and Barnard 1978).

Currently, the occurrence of facultative thermogenesis within the Pythonidae remains unclear (Stahlschmidt and DeNardo 2010). This limitation compromises our understanding of the factors that have shaped its evolution within the pythons and, as a result, decreases the utility of python facultative endothermy in evaluating the driving forces behind the evolution of endothermy. More complete knowledge of the existence of facultative endothermy among pythons would provide for a better understanding of the relative importance of phylogenetic constraints, morphological limitations, and environmental conditions on the existence of facultative endothermy, and thus provide insight into driving forces (and constraints) of endothermic capability in general.

The Pythonidae is divided between two primary phylo-genetic clades, an Afro-Asian clade with two major line-ages and an Indo-Australian clade with eight major lineages (Reynolds et al. 2014; for recent review, see Barker et al. 2015). The two species with conrmed facultative thermo-genesis are in the different cladesP. molurus within the Afro-Asian group and M. spilota within the Indo-Australian group. Conversely, both primary clades are also known to contain species where the absence of facultative thermo-genesis has been conrmedthe rock python (P. sebae) (Vinegar et al. 1970) and the ball python (P. regius) (Ellis and Chappell 1987) within the Afro-Asian clade and the reticulated python (P. reticulatus) (Vinegar et al. 1970), the water python (Liasis fuscus) (Stahlschmidt et al. 2012), and the Childrens python (Antaresia childreni) (Stahlschmidt and DeNardo 2009) in the Indo-Australian clade (Fig. 1). Brooding metabolic data are currently absent for all other species, including no representation from four of the ten major lineages.

The aim of this study was to assess the presence of brooding facultative thermogenesis in four previously unstudied species of python, including representatives from two of the four lineages where no data existthe black-headed python (Aspidites melanocephalus), the woma (A. ramsayi), and the bismarck ringed python (Bothrochilus boa). The addition of these species provides insight into possible phylogenetic inuence on endothermic capability. We also evaluated two other pythons. The jungle carpet python (M. spilota cheynei) is a member of the same

species as the diamond python (M. s. spilota) and the southwestern carpet python (M. s. imbricata), both of which have been shown to use facultative thermogenesis (Slip and Shine 1988; Pearson et al. 2003). Morelia spilota is widely distributed throughout Australia and thus experiences a great range with considerable variation in environmental temperature. Morelia s. spilota and M. s. imbricata are both from high latitudes, while M. s. cheynei is more tropical in distribution. Examining M. s. cheynei provides insight into environmental inuences on facultative thermogenesis. Lastly, we examined the ball pythons (Python regius) to clarify somewhat ambiguous prior results from this species. Together, these species combined with the previous species studied provide a considerable variation in adult size and thus may reveal any inuence that size may have on the use of endothermy.

Materials and methods

Animals

Over two breeding seasons (2007 and 2008), data were collected from 18 brooding females representing six species of python: 3 A. melanocephalus, 3 A. ramsayi, 2 B. boa, 3 M. spilota cheynei, 1 M. viridis, and 6 P. regius. Snakes were either borrowed from private breeders and housed at Arizona State University (ASU) during the two-year period or were part of a long-term captive breeding colony at ASU.

Animals were housed individually in temperature-controlled rooms (27 1 C) under a 12:12 h photoperiod

with supplemental heat provided at one of the cage by a subsurface heating element (Flexwatt, Flexwatt Corp., Wareham, MA, USA). During the non-breeding season (June to November), animals were fed weekly and provided water ad libitum. In November, animals underwent a 6-week cooling period in which we turned off the sub-surface heating elements and ceased feeding. In January, subsurface heating and feeding resumed. After 2 weeks, we began rotating males through the females cages. In February, we began weekly ultrasound scans (Concept/MCV, Dynamic Imaging, Livingston, Scotland) of females to assess their reproductive progress.

We weighed gravid females 1 week prior to oviposition. For three of the species (M. viridis, M. s. cheynei, P. regius), pre-oviposition females were placed in triple-ported, cylindrical containers (7.012.0 l), which we housed within a temperature-controlled walk-in environmental chamber (T = 30.5 0.3 C; 14:10 L:D photoperiod). The con

tainers were tightly sealed and supplied with humidied air (2040 ml/min; relative humidity (RH) = 8085 %).

For the other three species (A. melanocephalus, A. ramsayi, B. boa), because of their higher activity levels, we

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Fig. 1 Phylogeny of pythons (adapted from Reynolds et al. 2014) showing the presence (T) or absence (NT) of facultative thermo-genesis in pythons. The results from previous studies are shown in gray (see text for references), while results from the present study

are shown in black. The addition of B. boa, A. melanocephalus, A. ramsayi, M. viridis, and M. s. cheynei now provides data for species within eight of the ten major phylogenetic branches of Pythonidae. T/ NT for M. spilota represents differing results among three subspecies

placed similar brooding containers inside the pre-oviposition females cages. These containers had openings bored into the lids that allowed females to enter the container to oviposit. Following oviposition, the container holding the brooding female had its bored lid replaced with a solid lid and was then moved to the environmental chamber and provided air as described above. All females oviposited in the containers between April and June. The oviposition containers served as metabolic containers during the study.

Measurements

Rates of O2 consumption ( VO2; ml/h) and CO2 production ( VCO2; ml/h) were collected from brooding females using

a ow-through respirometry system within 7 days of ovi-position. Supply air was scrubbed of CO2 and water (CDA 1112, PureGas, Broomeld, CO), then routed through a mass ow controller (Unit Instruments, Inc., Yorba Linda, CA, USA) before entering the metabolic chamber containing the brooding female. Efux air from the metabolic chamber sequentially passed through a hygrometer (R2300, Sable Systems, Las Vegas, NV, USA), a drying vial of CaSO4, a CO2 analyzer (LI-6252, Li-Cor Biosciences,

Lincoln, NE, USA), and an O2 analyzer (FC-1B, Sable Systems, Las Vegas, NV, USA). The entire system was plumbed with minimally hygroscopic tubing (Bev-A-Line IV, Cole-Parmer, Vernon Hills, IL, USA). Air ow was adjusted for each metabolic chamber to replace 99 % of its

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Table 1 Female and clutch metrics of six species of python

air in less than 20 min using the 99 % equilibration equation of Lasiewski et al. (1966). Flow rates ranged from 0.75 to 2.0 l/min. Gas analyzers were calibrated weekly, and data were recorded using a datalogger (23X, Campbell Scientic Instruments, Logan, UT, USA).

Females were acclimated for 12 h before beginning data collection. Steady-state metabolic data were collected from each female over 12 h at each of the two temperatures: 31.5 C which approximates optimal developmental temperature of pythons (Ross and Marzec 1990) and 25.5 C, a sub-optimal temperature at which P. molurus is known to generate substantive heat (Brashears and DeNardo 2013; Van Mierop and Barnard 1978).

We collected temperature data from the clutch (Tc), the metabolic chamber (Tn), and the environmental chamber (Te) using type-T thermocouples feeding into the 23X data-logger. We measured Tc by inserting a thermocouple into the center of the clutch through a sealed port in the oor of the metabolic chamber. Tn was measured using a thermocouple that passed through the inux port in the side of the chamber so that it protruded ~5 cm into the metabolic chamber. Te was measured by securing a thermocouple ~10 cm from the metabolic chamber.

After each 24-h trial (12 h acclimation and 12-h data collection) to collect brooding data, the female was temporarily separated from its clutch to collect clutch metabolic data at the same temperatures using closed respirometry to preserve the hydric state of the unprotected clutch. Single-ported, closed respirometry containers were air tight and ranged in size from 1.2 to 6.7 l. We used 140 ml plastic syringes to draw initial and nal gas samples, then dried the air using CaSO4 before passing it through an O2 analyzer (S-3A, Applied Electrochemistry, Inc., Sunnyvale, CA,

USA). Durations of the trials were adjusted to allow for sufcient oxygen consumption to occur. Oxygen consumption of the clutch (ml O2/h) was calculated as (O2 % nal

O2 % initial) X functional chamber volume/time. The VO2 and VCO2 were calculated for the brooding unit (female plus clutch) using the equations supplied by Walsberg and Hoffman (2006) by taking the 20 min interval with the lowest metabolic values. Taking the 20 min interval with the lowest metabolic values eliminated periods of female activity and was indicative of the average trial values. Female alone VO2 was calculated by subtracting clutch VO2 from the brooding unit VO2.

Statistics

We used a repeated measures Generalized Linear Model with temperature as a within-subjects factor (two levels: 25.5, 31.5 C), species as a between-subjects factor (six species), and mass as a covariate to test for differences in metabolic rate. Within-species comparisons of metabolic

rate at the two different temperatures were completed by applying a single post hoc paired T test. All statistics were performed in SPSS (SPSS Inc., Chicago, IL, USA). All values are presented as mean SEM.

Results

All females maintained a tight coil around their clutches during the experiment until the clutch was removed for determination of clutch metabolism. The coils of most females covered the majority of the side and dorsal surfaces of the clutch; however, female B. boa displayed an unusual coil in that they wrapped completely around their clutches, including the underside of the clutches. We did not observe twitching by any female during or after the experiment. Female mass, clutch size, and clutch mass are presented in Table 1.

Brooding metabolic rates varied considerably among species (F5, 12 = 4.47, p = 0.016; Table 2), and there was

a strong interaction effect between species and temperature (F5, 12 = 8.73, p = 0.001; Table 2). Within a species,

Species, n Female mass (g) Clutch size (eggs)

Clutch mass (g)

2116 5 627.51724 6 644.03157 7 648.5 Mean SEM 2332 427 6 1 640.0 6.4

Aspidites ramsayi, 3 950 9 348.0

1008 10 439.7866 2 384.2Mean SEM 942 41 10 1 390.6 26.7

Bothrochilus boa, 2 1563 18 351.0

1780 13 243.0 Mean SEM 1671 16 297.0

Morelia spilota cheynii, 3

Aspidites melanocephalus, 3

2100 16 636.02200 19 760.31422 11 479.5Mean SEM 1907 244 15 2 625.3 81.2

Morelia viridis, 1 1000 13 126.0 Python regius, 6 1017 5 323.7

641 3 121.7734 4 212.0934 4 241.21137 7 722.01593 6 303.2Mean SEM 1009 138 5 1 294.1 95.7

Female mass, clutch size, and clutch mass are presented for each individual in the experiment

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Table 2 Metabolic metrics insix species of python Species, n Q10 25.5 C

RER VO2 (ml/h) VO2 (ml/h/kg)

Aspidites melanocephalus, 3 4.8 0.7 28.0 13.21.2 0.6 50.8 29.51.4 0.7 38.8 12.3 Mean SEM 2.5 1.2 0.6 0.1 39.2 6.6 18.3 5.5

Aspidites ramsayi, 3 3.6 0.7 24.4 25.72.1 0.8 14.8 14.62.3 0.7 19.0 21.9 Mean SEM 2.7 0.5 0.7 0.1 19.4 2.8 20.8 3.2

Bothrochilus boa, 2 0.7 0.7 64.3 41.10.6 0.7 182.3 102.4 Mean 0.6 0.7 123.3 71.8 Morelia spilota cheynii, 3 1.0 0.8 122.1 58.13.8 0.7 66.5 30.25.5 0.7 25.1 17.7Mean SEM 3.4 1.3 0.7 0.1 71.2 28.1 35.3 12.0

Morelia viridis, 1 3.5 0.9 18.2 18.2 Python regius, 6 1.4 0.6 12.3 12.11.6 0.8 13.6 21.10.4 0.4 25.1 34.21.0 1.4 28.3 30.31.6 0.9 29.2 25.78.9 1.6 14.0 8.8Mean SEM 2.5 1.3 0.9 0.2 20.4 3.3 22.0 4.1

Species, n Q10 31.5 C

RER VO2 (ml/h) VO2 (ml/h/kg)

Aspidites melanocephalus, 3 0.8 71.5 33.80.8 56.1 32.51.3 47.8 15.1

Mean SEM 1.0 0.2 58.5 7.0 27.2 6.0

Aspidites ramsayi, 3 0.4 52.8 55.50.9 23.2 23.00.7 31.1 35.9

Mean SEM 0.7 0.1 35.7 8.8 38.1 9.4

Bothrochilus boa, 2 0.7 53.0 33.90.8 128.7 72.3 Mean 0.7 90.8 53.1 Morelia spilota cheynii, 3 0.7 125.3 59.60.7 148.8 67.60.8 69.7 49.0

Mean SEM 0.7 0.1 114.6 23.4 58.7 5.4

Morelia viridis, 1 0.7 38.7 38.7 Python regius, 6 1.1 15.0 14.70.9 17.9 27.91.1 13.8 18.81.8 28.0 30.00.7 38.2 33.60.7 51.8 32.5

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Table 2 continued Species, n Q10 31.5 C

RER VO2 (ml/h) VO2 (ml/h/kg)

Mean SEM 1.0 0.2 27.5 6.5 26.3 3.2

Q10 thermal sensitivity, RER respiratory exchange ratio, total VO2 (ml/h), and mass specic VO2 (ml/h/kg)

are presented for each female in the experiment. Clutch VO2 was subtracted from total VO2 prior to calculations

the mass-adjusted VO2 was signicantly increased at the higher temperature for A. melanocephalus [t (2) = 4.47,

p = 0.046], A. ramsayi [t (2) = 5.72, p = 0.029], and

M. s. spilota [t (2) = 4.56, p = 0.044]. Mean Q10 values

for these three species were 2.5 1.2, 2.7 0.5, and

3.4 1.3, respectively (Table 2). Similarly, the single

M. viridis increased its mass-adjusted VO2 from 18.2 to 38.7 ml/kg/h (Q10 = 3.5; Table 2). Python regius had sub

stantial intraspecic variation in mass-adjusted VO2 at each temperature, which was reected in the lack of signicant difference in mean mass-adjusted VO2 between the two temperatures [t (5) = 2.23, p = 0.155; Table 2]. Python

regius had a mean Q10 value of 2.5 1.3 (Table 2). In con

trast to the other species, the mass-adjusted VO2 of both female B. boa decreased at the higher temperature, from 41.1 to 102.4 ml/kg/h at 25.5 C to 33.9 and 72.3 ml/kg/h at 31.5 C, respectively (Q10 = 0.8 and 0.6; Table 2).

We were able to collect temperature data from all species except B. boa (Table 3). Due to the unusual nature of their coiling, it was impossible to insert thermocouples into their clutches. Additionally, these females are extremely sensitive to any disturbance of their nesting environment and quickly abandon their clutches if disturbed. There were no signicant differences between Tc and Te within any of the other ve species (Table 3). Calculated temperature differentials from these ve species showed that T was not signicantly different from zero (Table 3).

Discussion

With the addition of our brooding metabolic rate measurements in A. melanocephalus, A. ramsayi, B. boa, M. s. cheynei, and M. viridis, there now exist data on facultative thermogenesis for representatives within eight of the ten branches of Pythonidae. While our study was limited by low sample size, the lack of comparative data on brooding python metabolic rates and the difculty in obtaining reproductive animals for experimental purposes make the data presented here valuable in furthering the understanding of what factors inuence the existence of facultative endothermy among pythons. Based on both metabolic (Table 2) and thermal (Table 3) assessments, our results strongly suggest that ve of the species are not thermogenic during brooding. While

the data from B. boa are less conclusive, in general, our results in combination with previous studies strengthen the growing realization that facultative thermogenesis among pythons is not as widespread as previously thought.

The higher metabolic rate at 25 C compared to 31.5 C in B. boa reects a pattern seen in facultatively endothermic species (Vinegar et al. 1970; Ellis and Chappell 1987). Unfortunately, we were unable to collect temperature data from the clutches of B. boa to denitely demonstrate a temperature differential between the clutch and the chamber. No twitches were observed at any time during reproduction in either female, but this does not necessarily imply that heat was not produced muscularly or metabolically. Muscle contractions that produce heat do not necessarily produce visible muscle tremors (e.g., birds) (Hohtola 2004). Regardless, despite being higher at 25.5 C, metabolism of B. boa was quite limited, and the resulting low power production (brooding max = 1 mW/kg at 25.5 C assum

ing lipid catabolism) seems insufcient to sustain a temperature differential between the clutch and chamber environment. In comparison, both facultatively thermogenic species, P. m. bivittatus and M. spilota, have a calculated maximum power production of approximately 1 W/kg (Van Mierop and Barnard 1978; Harlow and Grigg 1984). As a result of the low proportional increase in metabolic rate at 25 C as well as the low power output relative to that of species with effective brooding thermogenesis, we suspect that the heat production by B. boa would have little effect on the developmental environment unless the nest was extremely well insulated.

Work by Ellis and Chappell (1987) demonstrated that, unlike non-reproductive females, brooding P. regius have a VO2 that is insensitive to temperature (i.e., Q10 not signicantly different from 0) and, as a result, the metabolic rate at lower temperature is 34 times higher in brooding compared to non-reproductive females (Ellis and Chappell 1987). Our data from P. regius support this previous work in that our calculated mean Q10 value for P. regius, while relatively high at 2.5 1.3, was also not signicantly dif

ferent from zero. The high mean value was largely due to the unusually high thermal sensitivity of a single female (Table 2). With this female excluded, the mean Q10 for this species would be 1.2 0.2, matching the previous

nding that this species is metabolically uncoupled from

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Table 3 Temperature metrics in six species of python

Species, n 25.5 C

Te (C) Tn (C) Tc (C) T (Tc Te)

Aspidites melanocephalus, 3 25.5 25.7 25.5 0.07

26.3 26.3 26.2 0.08

25.7 25.4 25.7 0.07

Mean SEM 25.8 0.2 25.8 0.3 25.8 0.2 0.1 0.3

Aspidites ramsayi, 3 26.1 27.1 26.2 0.1725.4 25.8 26.6 1.1925.4 24.9 25.5 0.04 Mean SEM 25.6 0.2 26.0 0.6 26.1 0.3 0.5 0.6

Morelia spilota cheynei, 3 25.5 25.9 25.6 0.0725.8 26.3 26.0 0.2025.5 25.7 25.3 0.67 Mean SEM 25.6 0.1 26.0 0.2 25.6 0.2 0.3 0.2

Morelia viridis, 1 26.0 26.0 26.3 0.27 Python regius, 6 24.4 25.7 26.1 1.7726.3 25.7 25.8 0.47

26.3 26.1 26.1 0.20

25.9 26.0 26.4 0.4526.2 25.8 25.7 0.59

25.0 25.8 25.3 0.32 Mean SEM 25.7 0.3 25.9 0.1 25.9 0.1 0.2 0.4

Species, n 31.5 C

Te (C) Tn (C) Tc (C) T (Tc Te)

Aspidites melanocephalus, 3 31.9 31.9 31.6 0.28

32.4 32.2 32.2 0.18

31.5 32.3 31.3 0.14

Mean SEM 31.9 0.3 32.1 0.1 31.7 0.3 0.2 0.3

Aspidites ramsayi, 3 32.9 31.5 32.2 0.69

31.1 33.9 32.7 1.5631.4 33.9 32.3 0.86 Mean SEM 31.8 0.6 33.1 0.8 32.4 0.1 0.6 0.8

Morelia spilota cheynii, 3 31.3 31.9 31.6 0.3131.6 31.7 31.7 0.0831.3 31.6 31.4 0.10 Mean SEM 31.4 0.1 31.7 0.1 31.6 0.1 0.2 0.1

Morelia viridis, 1 31.8 31.4 31.9 0.13 Python regius, 6 31.3 31.5 31.7 0.4131.3 31.4 31.3 0.0931.2 31.3 31.4 0.1731.5 31.8 31.5 0.02

31.9 31.3 31.9 0.08

31.8 32.5 32.1 0.33 Mean SEM 31.5 0.1 31.6 0.2 31.7 0.1 0.2 0.2

Te environmental temperature, Tn metabolic chamber temperature, Tc clutch temperature, and T are pre

sented for each female in the experiment

temperature during brooding (Ellis and Chappell 1987). We also found no evidence that female P. regius are able to sustain a temperature differential (Table 3).

The value of limited but higher than expected metabolic rate at lower temperatures for brooding B. boa and P. regius is currently uncertain, and would require further study. In

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both Ellis and Chappell (1987) and our studies, the females brooded in an environment where there was considerable air ow through the brooding chamber to enable respiro-metric measurements. The two species (P. molurus and M. spilota) with demonstrated dramatic increases in metabolic rate at cooler temperatures are both known to brood on the surface (Slip and Shine 1988; Ramesh and Bhupathy 2010). However, P. regius is known to brood in burrows and the brooding location for B. boa is unknown. If B. boa also broods in a more insulated environment, the less impressive metabolic response of these two species to low temperatures may be ample to enhance the thermal conditions of development under natural conditions. Future studies should look at the impact of insulation on power demands of brooding female pythons in thermoregulating the developmental environment of their offspring.

Facultative thermogenesis does not have a clear phylogenetic signal in Pythonidae. Of the now 11 species of pythons that have been examined for facultative thermo-genesis, only two have been shown to have considerable heat production, two others have shown atypical temperature response curves suggestive of limited thermogenic potential, and seven fail to show any indication of thermo-genesis (Fig. 1). Thus, we conclude that strict ectothermy, not facultative thermogenesis, may be the norm for brooding pythons. Furthermore, the occurrence of facultative thermogenesis, whether pronounced or limited, is phylo-genetically widely dispersed among the family. Both the Afro-Asian and Indo-Australian lineages have species that have either pronounced thermogenesis, limited thermogenesis, or no detectable thermogenesis (Fig. 1). Given current data, it is not possible to determine whether facultative thermogenesis during brooding has evolved multiple times or whether it has been lost multiple times within the Pythonidae. However, the lack of a phylogenetic pattern to the presence and absence of thermogenesis within the pythons suggests a lack of any phylogenetic constraint on thermo-genesis within the group.

Size appears to act as a limiting factor in thermogenesis. To sustain the energetic demands of an elevated metabolism, the female must be able to possess considerable energy storage to support thermogenesis after investing 30 % or more of its body mass into egg production (Angilletta and Sears 2003). Additionally, larger size provides greater energetic potential (Shine 1992; Aubret et al. 2002) as well as greater clutch insulation. Thus, it is reasonable to suspect that small or thin species could not be facultatively thermogenic during brooding. This is supported by the fact that neither A. childreni, a member of a genus of small species, nor M. viridis, a thin arboreal species, are thermogenic (Stahlschmidt and DeNardo 2009; and this paper, respectively). However, large size alone does not guarantee facultative thermogenesis by females. Python reticulatus and P.

sebae are two of the three largest python species, yet both of them are non-thermogenic during brooding (Vinegar et al. 1970).

Geographical range appears to be a stronger predictor of facultative thermogenesis in pythons, but further analyses are necessary to support this preliminary conclusion. While large size is likely necessary to support facultative endothermy, cooler environments are likely necessary for the developmental advantages to offset the energetic costs. The three species that express pronounced facultative thermogenesis, P. molurus, P. bivittatus, and M. spilota, have distributions at the northern and southern latitudinal limits, respectively, of the family (Vinegar et al. 1970; Slip and Shine 1988). Facultative thermogenesis also appears linked to geographic distribution across the subspecies of Morelia spilota. While its presence has been demonstrated in M. s. spilota and M. s. imbricata, we show that M. s. cheynei is not facultatively thermogenic. Morelia s. spilota and M. s. imbricata reside in southeastern and southwestern Australia, respectively, and thus occur at the southern latitudinal extreme of pythons. However, M. s. cheynei lives in the sub-tropical northeast part of the continent (Wilson and Swan 2008). Because body size and shape are similar among the three subspecies (in fact, M. s. spilota is relatively arboreal and slimmer than the other two sub-species), it is likely that the lack of facultative thermogenesis in M. s. cheynei is more a reection of the relatively warm climate it inhabits more so than an inherent physiological or morphological limitation preventing the evolution of facultative thermogenesis in this race.

The question of whether facultative endothermy is ancestral to Pythonidae and has been maintained in large-bodied species inhabiting cooler climates, or whether it has evolved separately in multiple lineages is beyond the scope of this study. However, the high within-species metabolic variation, particularly within P. regius (Table 2), in the absence of detectable heat production (Table 3), is further evidence that metabolic rate may be particularly labile within Pythonidae. Several python species have been shown to increase metabolic rate 10- to 15-fold when digesting a meal (Secor and Diamond 1995; Ott and Secor 2007). Such variation could have provided the substrate for the evolution of facultative thermogenesis in species where the female can sustain the energetic costs and considerable developmental benets can be realized because of environmental conditions.

Finally, the existence of facultative thermogenesis in pythons has been used to support the reproductive model of the evolution of endothermy, whereby the initial driving force for the evolution of endothermy was the benets that heat production provided to the developmental environment of offspring (Farmer 2000). However, it has been pointed out that the substantial costs of thermogenesis

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may be prohibitive (Angilletta and Sears 2003). The rarity of python facultative thermogenesis supports this caution, but as yet there has been no consideration of strategies brooding females use to reduce these costs. Brooding females can reduce thermal conductance and thus energy expenditures via the selection of thermally favorable nest sites (Shine and Harlow 1996) and can supplement thermo-genesis with heat acquired through basking (Slip and Shine 1988). These factors need to be quantitatively evaluated if python facultative thermogenesis is to be useful in discussions regarding the evolution of endothermy.

Acknowledgments This study was funded by the National Science Foundation, IOS-0543979 to DFD. All procedures performed in this study were in accordance with the ethical standards of the Institutional Animal Care and Use Committee at Arizona State University.

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Facultative thermogenesis during brooding is not the norm among pythons

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