skink report.doc (11)

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Zoology; Professional Training YearInvestigation into the role of conspecific attraction in communal nesting behaviour of Australian skink sub-species Cryptoblepharus pulcher pulcher1218846

Vocational supervisor: Simon Clulow

Life Sciences, University of Newcastle, NSW, Australia

1218846

Stewart Wallis

Zoology

Investigating the role of conspecific attraction in communal nesting of Australian skink sub-species Cryptoblepharus

pulcher pulcher

Word Count: 4,851

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Part 1 – Overall PTY Experience

My overall experience participating in a research project at the University of Newcastle has been incredibly positive and enjoyable. I feel it has not only been a valuable insight into the dynamics of a true laboratory-style research environment, but also equipped me with an array of novel skills that would be difficult to acquire in any other setting. Primarily, I feel I have developed and matured on a personal level. For the first time, I have had the task of setting-up, conducting, and maintaining a project by myself on a large scale, which has encouraged me to be more responsible and thorough in my work, with greater attention to detail as any mistakes would impact me and my findings directly. From the first week of my PTY placement, my supervisor has managed to achieve a good balance of helpful guidance and encouragement of independence. Although this freedom was initially unfamiliar and difficult to get used to, it became much easier as the project progressed to trust my initiative and take the lead, approaching my supervisor only when it was necessary. As a result of this, I have learnt to be more aware of my own abilities, and trust that as the primary researcher, I have a perfectly clear notion of what is going on.

I have been unbelievably fortunate with the opportunities afforded to me during this placement within my project and beyond; from the anatomical examination of a Komodo dragon to the many remote locations holding an abundance of Australian reptiles that were available to me. Conducting this project in a completely different country has allowed me to experience research from an alternative perspective, as well as draw universal similarities across different scientific communities, expanding my wisdom and appreciation for Zoology.

My time in the laboratory was distributed across various roles. There was a large field work component to begin with which I enjoyed immensely. Coupled with that was lab work, which compiled most of my time overall. The specimens we captured needed daily monitoring and care, ensuring variables such as temperature and humidity were kept constant, to keep their environment as controlled as possible. At the busiest part of the experiment, my attendance was 6-7 days a week, which I gladly did, as I was enjoying the work, and looked forward to carrying it out. Alongside this, I took my time to get involved in other research projects around the lab, which enlarged my social circle and educated me on other similar pioneering herpetology. I took part in visiting other field sites, aiding my peers in frog surveys and capturing, which not only helped their research but also opened my eyes to the discipline and admirable dedication these people have to their work. Everyone I met in the laboratory was very friendly and accommodating, taking interest in my project and assisting me when I asked. It was a fantastic experience and I would encourage any other student to seriously consider undertaking a PTY.

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Part 2 – Written Report

Abstract - It was the purpose of this experiment to determine if conspecific attraction plays a role in communal nesting of Australian skink subspecies, Cryptoblepharus pulcher pulcher. To do this, 100 gravid females were caught and 10 were housed in 10 separate, but identical, polyethylene tubs, within which were 10 identical nest sites. The conditions of all 10 tubs and nests were kept constant throughout the investigation, to remove any other cue that could encourage laying preference in any nest. In the 5 control tubs (A-E), once an egg was laid, it was removed to eliminate conspecific attraction as a cue. In the 5 experimental tubs (F-J), any eggs laid were left untouched. It was hypothesised that in tubs A-E, random nesting patterns would occur as there was no cue to trigger conspecific attraction and therefore communal nesting. Tubs F-J would show communal nesting patterns, and multiple clutches would be deposited in only a few nest sites. Communal nesting was accepted in nests that showed 2 or more clutches laid, and solitary nesting was accepted in nests that showed a single clutch laid. The results show that in tubs A-E, there were 3 instances of communal laying, and 4 instances of solitary laying. Tubs F-J experienced 6 instances of communal laying and 4 instances of solitary laying. As there seemed to be no significant preference towards communal nesting behaviour in the experimental tubs, we concluded that random nesting patterns occurred consistently, therefore disproving part (b) of the hypothesis. Only 26 out of the 100 specimens were actually gravid and laid eggs, 3 tubs had no instances of laying at all and were invalid for this reason, so it is with caution that we accept these results due to inadequate sample sizes. Reasons behind the evolutionary advantages of communal nesting are also explored.

Introduction – “Many reptiles lay their eggs together in the most suitable sites, but it is difficult to account for the colonial nesting habit...without assuming the nesting females are in some way attracted by eggs of their own species” (Noble and Masom 1933)

It is beneficial for an individual of a species to locate or construct some kind of nest during the reproduction process. Whether this nest functions as a protective barrier, a means of crypsis or thermoregulation, having a structure within which to lay ones eggs or raise off spring, maximises the chances of survival for the vulnerable, developing generation (Collias, N.E. and Collias, E.C., 1984). However, some species across a variety of animal classes, have exhibited communal nesting behaviours (also referred to as communal egg-laying, joint nesting, colonial egg-laying and egg dumping). That is, the non-incidental deposition of eggs at a shared nest cavity by two or more conspecifics (Alfonso et al., 2012). Animal egg aggregations have received very little attention, and have been a poorly understood example of social reproduction (Doody, Freedberg, and Keogh, 2009). However, it is known that communal nesting is taxonomically widespread across a number of organisms within the animal kingdom, ranging from birds, frogs, salamanders, fish,

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Zoology; Professional Training YearInvestigation into the role of conspecific attraction in communal nesting behaviour of Australian skink sub-species Cryptoblepharus pulcher pulcher1218846molluscs, dragonflies, mayflies, caddisflies, bees and butterflies to name a few examples (Doody, Freedberg, and Keogh, 2009). Research has shown communal nesting to be a well-established and conserved behaviour, the antiquity and perseverance of communally laid eggs is evident from their occurrence amongst some dinosaurs (Horner, 1982). Evans and Hook (1992) discovered communal nesting in the digger wasp (Cerceris australis). Nests were usually occupied by several females, some of which are ‘provisioners’, bringing in food day after day and each time, leaving the nest usually after only a few seconds, and others were ‘non-provisioners’, leaving the nest for a short period just once a day and returning without prey. Swanson (2004) discovered communal nesting in the apple murex snail (Phyllonotus pomum), stating that ‘communal oviposition is common among gastropod molluscs’. The remarkable diversity in the evolutionary histories of these taxonomic groups raises huge difficulty in determining any generality for colonial nesting. Communal nesting is not exclusive to oviparous organisms, Ebensperger (2004), found that female Degus (Octodon degus) formed stable associations of > 2-4 female individuals, all of whom shared a single nest at night, within which they birthed and reared their offspring. However for the purpose of this experiment, the focus of the surrounding research will be on oviparous organisms as C. pulcher pulcher are egg-laying.

The phenomenon of communal nesting is easily explained in birds; baby birds generally require a considerable amount of parental care after they are born (Doody, Freedberg, and Keogh, 2009). By nesting together, adult birds can share the burden of feeding with ‘helpers’ and protect the young (Skutch, 1961), giving a plausible advantage to communal nesting. A five year study of the Florida scrub jay (Aphelocoma coerulescens) revealed that Florida scrub jay helpers participate in territory and nest defence by mobbing predators, and in certain phases of care of the young. They fed nestlings and fledglings, and defended the nest. They also removed faecal sacs (Woolfenden, 1975). On the contrary, reptiles often abandon their eggs after laying; with parental care found in only 20% of salamanders, 6% of frogs, 3% of snakes and 1% of lizards (Doody, Freedberg, and Keogh, 2009), so for the majority of reptile groups, distributing parental duties cannot be the reason behind their communal nesting. Notably, communal nesting has been discovered in species with attending mothers, suggesting both are not mutually exclusive (Doody, Freedberg and Keogh 2009). To date, communal nesting is found in 345 lizard species, as well as many snake and alligator species (Doody, Freedberg, and Keogh, 2009), whilst a large percentage of reptile species still remain unaccounted for. Swain and Smith (1978) discovered a nest of 118 Coluber constrictor eggs and shells under large rocks at a construction site. They reported this may be due to severe local limitation of suitable nest sites, as well as a strong social affinity of some species. The phenomenon has been recorded in Opheodrys aestivus (Palmer and Braswell, 1976), and an egg laying aggregation in Oregon of 294 eggs belonging to 4 separate snake species was discovered with more than half the eggs within a 1.8 m area (Brodie, Nussbaum, and Storm, 1969).

The reasons for reptile aggregation during oviposition can be split into two theories. Firstly, the ‘constraint’ theory (or saturated hypothesis theory) proposes that gravid

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Zoology; Professional Training YearInvestigation into the role of conspecific attraction in communal nesting behaviour of Australian skink sub-species Cryptoblepharus pulcher pulcher1218846females are mutually attracted to environmental conditions that have limited availability (Graves and Duvall, 1995). In other words, reptiles communally nest simply because good nesting spots are scarce in reptile habitats; therefore communal nesting is a necessity and not an evolutionary advantage as with birds. There is also growing evidence to suggest an evolutionary benefit to laying communally for reptiles, despite the lack of parental care. The ‘adaptation’ theories suggest there are fitness benefits to laying such large communal clusters, either to the developing offspring, or the mothers (Radder and Shine, 2007). The latter is based on two important observations; 1) communal clutches are often seen in areas with an abundance of useable nest sites, and 2) ‘conspecific cueing’ has been observed in snakes, lizards and frogs (Doody, Freedberg and Keogh 2009). Conspecific cueing occurs when an organism obtains information on the habitat quality from the distribution of conspecifics. Consequently, when conspecific cueing is used, organisms should prefer to nest in areas with high occupancy.

In this experiment, it was hypothesised that separate groups of gravid C. pulcher pulcher, would deposit eggs in different patterns (random/clustered) depending on what treatment the eggs were given once laid (removed/left untouched).It was therefore the purpose of this experiment to investigate the tendency for reptiles to aggregate during oviposition, and what the role of conspecific attraction may be during the communal nesting process. We explored if the presence or absence of other eggs encourages Cryptoblepharus pulcher pulcher to lay in a clumped or random distribution. Although it is unfortunate that this has yet to be achieved, an important pilot study has been performed with this experiment, and will allow future replications to be much easier, not only for C. pulcher pulcher, but also other small skink species.

Materials and Methods –

Field work; Capture of the specimens

The elegant snake-eyed skink (Cryptoblepharus pulcher pulcher) is a small oviparous lizard species of the family Scincidae that is distributed along the arid southern coast of Western Australia and South Australia, as well as the coast, ranges and slopes of mid-Eastern Australia (Cogger, 2014). Being closely related to the wall-skink (Sternfield, 1918), C. pulcher pulcher commonly inhabits buildings and other human structures (Cogger, 2014). There were a total of three field sites from which adult gravid C. pulcher pulcher were collected. The first was the south-east facing stone wall of Camperdown Memorial Rest Park in Newtown, Sydney (-33.894838, 151.178441). The second was an unnamed north-facing stone wall in Newcastle, Australia (-32.927274, 151.775219). The third was the unnamed north facing stone wall running parallel to Darley road in Manly (-33.8026517, 151.289601). All sites were urban and humid climate, vertically sloped and highly residential, situated within the cities themselves and surrounded by roads. The skinks would bask on the walls at mid-morning hours through to early afternoon, during which time capturing would take place via hand by a team of 2-4 volunteers. To maximise the chances of catching gravid females, the capture process took place during the peak of C. pulcher pulcher’s breeding season, which was early December

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Zoology; Professional Training YearInvestigation into the role of conspecific attraction in communal nesting behaviour of Australian skink sub-species Cryptoblepharus pulcher pulcher1218846to late February. Being small and highly agile, the most efficient way to catch C. pulcher pulcher would be to approach slowly at first then rapidly place a flat palm on top of the skink, pinning it to the wall. It is important to immobilise the body of the skink and not trap the tail, as they possess an effective anti-predatory ability to shed their tails and escape. The specimens were collected daily in groups of 10-12.

Laboratory set-up and housing of the specimens

Once collected, specimens were housed in groups of 10, each group was confined within 10 separate but identical polyethylene tubs (1070mm x 340mm), labelled A-J which contained newspaper to absorb excreta and a centrally mounted infrared heat lamp to allow natural reptile thermoregulation. The tubs were also circular in shape, to eliminate corner effect. 10 identical nest sites were also positioned in a clockwise layout, labelled 1-10, and all equidistant (15cm) from the heat lamp. It was the primary aim during the laboratory set-up process, that C. pulcher pulcher’s natural nesting environment was replicated closely as possible. Little is known about the specific nesting requirements of different skink species, but from our own observations, it was decided that C. pulcher pulcher preferred small, dark, humid cracks and crevices, preferably on a vertical, sun facing stone-surface. Unfortunately it was not practical to conduct this experiment outdoors in direct sunlight, but after a range prototypes, it appeared C. pulcher pulcher had an affinity towards one in particular.

Figure 1a) A schematic diagram of the final prototype for all 100 nests. Each nest was equally distanced from the centrally mounted heat bulb, to ensure the temperature in each nest was unanimous. Petri-dishes were refilled daily to allow a continuous absorption of moisture into the nest cavity, maintaining the humidity at 80-85%.

2.5cm1.5cm

6.5cm15cm

Tuppaware lid containing a thin layer of reptile vermiculite

Wooden block

Uniformly folded tissue paper

Petri-dish filled daily with water

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Zoology; Professional Training YearInvestigation into the role of conspecific attraction in communal nesting behaviour of Australian skink sub-species Cryptoblepharus pulcher pulcher1218846The nests were compiled from identical, 15 cm x 6.5 cm, upside down, U-shaped wooden blocks, creating a small dark tunnel (2.5 cm x 1.5 cm) within which the specimens could crawl inside to deposit their eggs. Each nest rested on a plastic Tupperware lid holding reptilian vermiculite. This acted as a moisture-holding substrate to maintain humidity. It also reduced potential olfactory cues that other substrates such as soil could give off, and in turn, encourage nest-specific laying. A wick system was then used to maintain the humidity of each nest at 80-85%, with one end of uniformly folded tissue paper placed in the nest, and the other in a small petri dish of water that was refilled daily (Fig. 1a). To measure humidity and temperature, every third day, three random nests from each tub were selected using a random number generator (www.random.org), and recordings were taken using a Vaisala HM70 hand-held temperature and humidity meter. Specimens were fed crickets supplied by Pisces Enterprises three times a week. Egg checks were performed daily by gently lifting each wooden block with minimal disturbance, and each Tupperware lid, taking extra care to check for any eggs under the moist tissue wicks or outside the nests. The 10 tubs (A-J) were split into two sub-groups, A-E were the experimental tubs, and all eggs laid in any of the nests in these tubs were removed with a small spatula to eliminate cues, and placed immediatley inside a reptile egg incubator. Tubs F-J acted as control tubs, and any eggs laid in these tubs were left untouched, to encourage other gravid mothers to lay alongside them.

Measuring Humidity and Temperature;

To measure humidity and temperature, every third day, three random nests from each tub were selected using a random number generator (www.random.org), and recordings were taken using a Vaisala HM70 hand-held temperature and humidity meter. The end of the probe was slowly inserted into the nest cavity, allowing any specimens inside to escape with minimal stress. The reading was then taken from the meter after it has stabilised and appeared to be unchanging, this varied from a matter of seconds to several minutes. During the measuring process, the tip of the meter was hovered above the vermiculite and did not touch the walls of the nest, to receive the most accurate reading of the empty space within the nest itself.

Results

Egg Collection;

Figure 2a) This table summarises all eggs retrieved during the investigation. No instances of laying occurred in tubs D, F and J, prompting their elimination from the results. The total number of clutches collected throughout the experiment was 26, containing a total of 38 individual eggs.

The total number of all C. pulcher pulcher eggs laid in both control and experimental tubs.

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Proportion of communal and solitary clutches laid (%)

Solitary Communal

Specimens housed in control tubs A-E displayed 3 instances of communal nesting overall. 3 clutches were laid in nests B2, B6 and E10 (Fig. 2a). There were a total of 4 instances whereby 1 clutch was solitarily laid in separate nesting cavities, A1, A6, A7 and C8. Figure 2b indicates solitary nesting accounts for 57.1% of all clutches laid in the control tubs. These results do not support part (a) of the original hypothesis that specimens in control tubs A-E will exhibit random nesting patterns in a response to eggs being immediately removed once laid. Comparatively, experimental tubs F-J exhibited 6 instances of communal nesting in nests G3, G5, G6, H3, H9 and I5 and 4 clutches being laid in separate nest cavities; G1, G7, H1 and I4 (Fig. 2a). A total of 26 clutches were laid overall, which is considerably less than the expected 100 clutches (one clutch per gravid female). With a 26% lay rate there is not a sufficient data set to accurately draw any assumptions regarding role of conspecific attraction in communal nesting of C. pulcher pulcher. Pearsons Chi-squared tests revealed a p-value of 0.14, revealing no significant difference between solitary and communal nesting and treatment of the eggs. However as previously mentioned, an insufficient dataset retrieved from this experiment prompts difficulty is accepting validty with these results.

Temperature and Humidity monitoring;

Figure 2b) What percentage of all laid clutches were both solitary and communal was calculated to reveal which nesting behaviour was carried out preferentially amongst the specimens.

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The calculated mean humidity of each tub over time

The calculated mean temperatures of each tub over timeFigure 2a) The temperatures of three randomly selected nests in each tub were measured and the means calculated. This average for each tub was carried out on a three-day basis. The change in the mean nest temperature was plotted over the course of the investigation to reveal any notable thermal changes, and to ensure temperature remained constant enough to not directly affect the nest choice of the females.

Figure 2b)mean calculated. This average for each tub was carried out on a three-day basis, alongside temperature. The change in the mean nest humidity was also plotted over the course of the investigation to reveal any notable changes in humidity, and to ensure humidity remained constant enough to not directly affect the nest choice of the females.

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Zoology; Professional Training YearInvestigation into the role of conspecific attraction in communal nesting behaviour of Australian skink sub-species Cryptoblepharus pulcher pulcher1218846The results from monitoring humidity show two distinct peaks on 20/01/2016 and 07/02/2016. It is difficult to determine if daily forecast impacted the conditions of the tubs overall as forecast was not documented at the time. Although unlikely, as the experiment was carried out indoors, the overall surge in humidity on 07/02/2016, is likely due to some external factor. Although individual differences in the temperature and humidity of each nest may have impacted laying preference, there is an insufficient amount of laying to draw conclusions from this data.

Tub Highest Mean Temperature (°C)

Lowest Mean Temperature (°C)

Thermal Range (°C)

A 24 26.1 2.1B 22.6 26.9 4.3C 24.4 26.9 2.5D 24.6 27.6 3E 24.2 28 3.8F 24.9 28 3.1G 25.3 27.4 2.1H 25.7 28 2.3I 24.6 27.7 3.1J 25.8 27.6 1.8

LS Means PlotFigure 2c) The highest and lowest mean temperatures taken from each tub from an average of three random nest temperatures reveals the thermal range within each tub. Tub B harboured the greatest thermal range of 4.3°C across the nests, and Tub J the lowest, 1.8°C.

Figure 2d) The calculated LS Means Plots for temperature and humidity (Fig 2e) show the variation of mean tub temperature and humidity over time. This was influenced by biotic factors such as climate.

Figure 2e










































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Double Effects tests

Effects tests revealed a greater variation in temperature across days than across tubs, (Fig. 3a, Fig 3b). This indicates the attempts to control the microhabitat of the nests and keep them consistent were commendable despite a changing daily climate. However, there is a <0.0001 Prob > F, meaning there is a significant difference in the temperature and humidity throughout the investigation that could have influenced the females choice in choosing sites of egg deposition.

More evidence for this appears upon a closer look at Figure 1b. An interesting duplicated pattern emerges when temperature is compared across tubs instead of within the tubs themselves. There is a clear mirroring pattern displayed unanimously resulting in general temperature peaks and dips over time. This suggests an external

factor controlling temperature increase, most noticeable on 14/01/16 and decline, 07/02/16. To conclude, changing climatic conditions resulted in shifting temperatures and humidity across both experimental and control tubs. This was a significant enough that it could have impacted the skinks behaviour during the nest selection process, and created a preferential bias towards certain nests. However, due to a lack of sufficient data as a result of non-gravid specimens being captured by

Figure 3b) The calculated Effects test for humidity

Figure 3a) The calculated Effects tests for temperature

Fig 2f shows clearly Tubs A and B to be of much cooler temperatures overall. Tubs A and B were positioned closest to the window during the experiment, and as a result, were closer to outside and it appears more sensitive to climatic change.










































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Zoology; Professional Training YearInvestigation into the role of conspecific attraction in communal nesting behaviour of Australian skink sub-species Cryptoblepharus pulcher pulcher1218846mistake, it is not possible to draw any accurate conclusions until future replications with 100% gravid female capture can be analysed.

Discussion

Communal egg laying occurs in a vast array of reptile species, and has re-evolved and been lost many times during reptilian phylogeny (Radder and Shine, 2007). Thus, no singular hypothesis is likely to explain the functional significance of such a diverse and labile trait. It is clear that communal egg-laying is widely dispersed throughout lizard families, and has evolved independently multiple times (Doody,Freedberg and Keogh 2009).

It is virtually impossible to construct acceptable ancestral character states because a vast majority of information about communal egg-laying is absent. Herpetologists cannot accurately label a species “non-communally laying” when the eggs and nests are unknown. However it is possible to ascertain that the evolution of communal egg-laying started with many independent origins, which is consistent with the two main

Figure 3a) This diagram displays the distribution of communal nesting across lizard families. Families featured in bold represent the lizard families that are known to communally nest, and the following percentage is the known proportion of species that exhibit communal nesting

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Zoology; Professional Training YearInvestigation into the role of conspecific attraction in communal nesting behaviour of Australian skink sub-species Cryptoblepharus pulcher pulcher1218846groups of hypotheses of communal egg-laying mentioned earlier, constraint and adaptation. Surprisingly, very few studies have been done to test these hypotheses. More than 80% of eggs laid by Bassiana duperreyi (another scincid lizard, occupying the Brindabella range in south-eastern Australia) were found to be deposited communally. It was concluded that this high incidence reflected a clear preference in gravid mothers. Supporting this, laboratory findings showed nesting females oviposited in sites that contained cues from previously laid eggs (Radder and Shine 2007). The constraint hypothesis of communal egg laying is not supported by Radder and Shines laboratory work, because the frequency of communal oviposition was not affected by spatial and temporal variation in the availability of nest-sites. A study by Harris et al (1995) whereby the population density of female salamanders was manipulated, found there to be no effect on the number of instances of communal egg-laying. Although one would expect a higher instance of communal laying in a higher population density due to greater habitat saturation. Regarding the alternative, ‘adaptation’ theories, information passing through animal systems via social acquisition has been commonly observed, and animals often make critical decisions based on copying conspecifics (Pruett-Jones, 1992). It is, therefore, accurate to assume communal egg-laying has evolved as a result of ‘copying’ or ‘freeloading’ mothers expending no energy or time finding a nest, should the benefits outweigh the costs (Doody, Freedberg and Keogh 2009), leading to a maternal-benefits hypothesis. The nest building process can be a challenging task for reptiles. Some female lizards, may spend days constructing a nest that is suitable enough to deposit her eggs. During those days, she is not finding food or water, or basking, she is using energy and is more vulnerable to predation. Females can avoid these costs by simply laying eggs in a nest that someone else has gone to the trouble to build (Doody, Freedberg, and Keogh, 2009). There is a general overlying criticism with this approach however, in that they fail to explain why some reptile species do not construct nests at all, and when they do, in many cases they do not exhibit nest defence behaviour, prompting further questions into the accuracy of these maternal benefits.

Adaptive hypotheses of communal nesting surround various components of reptile behaviour in an attempt to explain reasoning behind this particular trait. Another approach suggests communal egg-laying and nesting to be of significant social importance (Graves and Duvall, 1995). Despite social interactions being less generally complex in reptiles than other tetrapods, remarkable social interaction has occured at reptilian communal nesting sites (Graves and Duvall 1995). C. pulcher pulchers social behaviour was not monitored in this experiment, although specimens were frequently observed interacting (chasing, basking together etc.) there was no indication this was a cause for communal nesting, and neither was there indication this didn’t just occur because the skinks were in a confined space. Alternatively, the kin selection hypothesis states that mainly relatives contribute to communal clutches (Tallamy, 1985). Females from the same maternal lineage will accumulate around the same nesting points, causing a clustering of related females. The resulting communal egg-laying is not so much a true survival adaptation but more likely a natal homing instinct (Graves and Duvall 1995). There is no current evidence

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Zoology; Professional Training YearInvestigation into the role of conspecific attraction in communal nesting behaviour of Australian skink sub-species Cryptoblepharus pulcher pulcher1218846supporting this idea in reptiles though, however. For future experiments, it could be beneficial to take DNA samples from each mother to determine amino acid sequences before laying, and house them with their closely related counterparts to investigate this. A much easier method to monitor relatedness in the skinks would be to house individuals caught from the same site together, assuming these animals would be more closely related than skinks from different sites.

Another suggested theory is the attack abatement theory. This proposes a predator is less likely to find a large group of many eggs than lots of little groups of eggs. By laying collectively, the dilution effect would predict an egg number increase per site, and reduce casualties due to predator satiation (Harris et al., 1995). The implication of predator satiation as an adaptive mechanism for communal nesting in reptiles would depend greatly on the size of the predator and the number of eggs destroyed. Larger vertebrate predators have been known to take entire clutches, whereas smaller invertebrate prey may take only a few. Nonetheless, little evidence exists to support this claim, with only one study by Brown and Duffy (1992) testing for predation rates on communal and solitary nests, and the results showing no preference towards nests of either type. There is evidence to suggest mothers use visual cues as indications of direct offspring fitness, and lay accordingly. The reproductive success-based hypothesis proposes that many gravid females will return to the same nest site continually each breeding season to deposit her eggs because they associate that site with successful offspring. According to Doody, Freedberg and Keoghs (2009) large literary review of reptile and amphibian communal egg laying, over one third of reviewed papers included egg shells present in communal nests from previous years, providing strong evidence for this theory. In addition to that, gravid common keelbacks in the lab preferentially deposited eggs in nests with shells from conspecifics over empty nests, prompting the authors to conclude successful previous hatching as a cue for communal egg laying. Finally the egg insulation hypothesis argues that communal egg laying may offer a thermal advantage in ectotherms, giving a plausible reason behind the evolution in this behaviour. Disappointingly, Radder and Shine (2007) found that thermal environments of communal and isolated nests did not actually differ at cold extremes. However, communal snake egg masses were an average of almost 5°C warmer than solitary clutches, when laid < 20km from a cold climate margin (Blouin-Demers, Weatherhead, and Row, 2004). Further incubation experiments revealed that warmer clutches provided by communal nests produced faster, larger and stronger hatchlings. A criticism of this approach is that even in warm, tropical climates, where insulation is relatively unnecessary; reptiles are known to lay communally. More analysis is needed into the environmental differences of different communal laying species before this hypothesis can be accepted. Temperature as a controlled variable was consistently measured throughout the C. pulcher pulcher experiment, by taking an average reading from three random nests on a tri-daily basis, with one of the key design features of the laboratory set up being maintenance of a constant temperature gradient across all nest sites. Given the opportunity to repeat this investigation, recording the temperature of all nests rather than three randomly selected nests would be advisable as this would produce a wealth of

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Zoology; Professional Training YearInvestigation into the role of conspecific attraction in communal nesting behaviour of Australian skink sub-species Cryptoblepharus pulcher pulcher1218846information on themal conditions and make for a more detailed analysis of temperature as a variable. All nests were positioned 15 cm from the heat lamp which removed thermal bias and ensured specimens wouldn’t favour a nest because it was a different temperature to the rest. However, not all tubs were positioned equidistant from the outdoors, and as Fig 2f has demonstrated, this could have affected the micro-climate of each tub. Notably, the thermal range within any tub did not exceed 4.3°C (Fig 2c), temperature as a variable was not kept perfectly constant and future replications of this experiment might invest in a sealed environment, or preferably, separate incubators to exercise greater control on temperature. It is not possible from our lack of data to ascertain if C. pulcher pulcher preferred warmer nests, although the one recorded instance of communal laying took place in Tub G which exhibited the joint lowest thermal range of 2.1°C in any tub where laying occurred, suggesting a smaller difference in temperature between nests resulted in females selecting a lay site based on conspecific cues.

It is important to note that none of these adaptive hypotheses are mutually exclusive, and it is likely that a combination or all are the true reason behind reptile communal laying. Neither one can explain this phenomenon if standing alone. There is sufficient evidence however to reveal the constraint hypothesis of habitat saturation to be inadequate in its description of communal nesting, and there are undeniable forces driving the evolution and maintenance of this behaviour across many animal groups. Further investigation is needed however to confirm the validity of each adaptive theory, and demonstrate communal nesting is not a result of a reduced number of adequate nesting sites.

Considering communal nesting from the opposing perspective, solitary nesting does occur ubiquitously alongside communal nesting not only in the same species, but in the same populations, suggesting survival benefits as well as costs to ovipositing alone. The purposeful or accidental ruination of eggs by other conspecifics is a huge cost, especially with high density populations. Alonso et al. (2002) discovered 20% of crocodile nests were destroyed by other nesting females over a period of 11 years. In addition to that, eggs that are unearthed or left exposed by other females could easily desiccate or be predated upon. Disease transmission can also pose a big problem for clutches laid communally, less so for hard or parchment shelled eggs laid by skinks such as C. pulcher pulcher, but still enough to be taken into consideration. During this experiment, the humidity was originally maintained at 90%-95%, based on an experiment with Lampropholis delicata by Doody and Paul (2013). This environment proved to be excessively moist for C. pulcher pulcher as eggs were frequently developing mould and perishing, hence the decision to lower the humidity to 80%-85%. But during this brief period, it showed transmission of the mould to other eggs within the nest was fast-moving and irreversible. Whether this mould acted as a deterrent for communal nesting, supporting the reproductive-success based hypothesis, is a fascinating direction to take this experiment in the future. In addition to providing healthy eggs as cues to trigger communal nesting, it would serve to provide damaged, desiccated or infected eggs as cues to deter communal laying.

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Zoology; Professional Training YearInvestigation into the role of conspecific attraction in communal nesting behaviour of Australian skink sub-species Cryptoblepharus pulcher pulcher1218846 The biggest and most obvious criticism of our experiment is the lack of useable data, which directly impacted our results as it was difficult to find enough instances of C. pulcher pulcher laying to work with. Collectively, only 26 clutches were laid by a group of 100 C. pulcher pulcher, which was the major shortcoming of the investigation. An unforeseen difficulty with the field work element was the identification of gravid females. The quickest and only conceivable way to do this was through visual cues, specifically, a distended abdomen indicating a gravid female. However, it was discovered that C. pulcher pulcher would harbour a distended abdomen when basking to increase their surface area, and after feeding, making them almost visually identical to gravid mothers. C. pulcher pulcher are a relatively small species of scincid lizard, with adults reaching a SVL of 41mm (Wilson and Swan, 2003), therefore distinguishing gravid females was an incredibly difficult task, and was the main contributing factor to our insufficient amount of data. During the set-up of the investigation, it was preferred that the 10 polyethylene tubs were to be kept in close proximity to one another, so the egg checks, watering, feeding and measurements could be completed in an efficient amount of time. Subsequently, the tubs took up a large amount of space, and therefore had to be positioned at different heights (on desks and on the floor), which could very easily have affected our experiment. Some tubs were also closer to windows and doors than others, leading to a lower average temperature on colder days.

Maintaining the nest humidity and temperature was a challenging task of paramount importance. Abiotic factors like humidity and temperature are well known to influence the oviposition-site selection of reptiles. Research shows a huge preference of egg laying reptiles towards more moist substrates, because of an increase in the phenotypic fitness of the offspring (Brown and Shine, 2004). Gravid common keelbacks (Tropidonophis mairii) showed a clear preference towards more moist substrates for egg deposition which significantly increased body size and muscular strength at hatching, a trait which is under strong positive selection for this species (Brown and Shine, 2004). Although we did exercise a high level of control on the substrate used for the nests (reptilian vermiculite), unfortunately it was not a natural substance that the specimens would normally encounter in the wild, and for this reason it may have affected our results. Controlling the substrate may not have been necessary; gravid (Tropidonophis mairii collected from tropical Australia were given a choice of potential nesting sites in captivity. Females selectively oviposited in sites containing empty eggshells rather than in control sites but failed to avoid the scent of a sympatric egg predator (Stegonotus cucullatus) in fact, eggshells of this taxon were just as effective as T. mairii eggs in attracting oviposition (Brown and Shine, 2005), suggesting olfactory cues bear little importance in the selection of nest sites, and the more important cues are purely visual. In retrospect, it could be advised that future replications of this experiment use naturally occurring soil with olfactory cues removed through a soil washing and drying procedure. It must also be noted that the C. pulcher pulcher captured were originally found on stone surfaces, therefore there could be a general preference towards no substrate and hard stone for oviposition; maintaining a high humidity in this environment would be much more difficult, however.

17

Zoology; Professional Training YearInvestigation into the role of conspecific attraction in communal nesting behaviour of Australian skink sub-species Cryptoblepharus pulcher pulcher1218846There is quite a deficient compendium surrounding communal nesting behaviours across reptile species, particularly lizards. With the vast majority of information targeting amphibians and snakes, this remains the only investigation into these behaviours of C. pulcher pulcher specifically. As discussed, there are many refinements this investigation would need for future replications to be conducted with greater accuracy, and generate a larger and more significant data set. Personally, I would be compelled to replicate slightly modified versions of this experiment across all Australian skink species, accommodating for different habitat, temperature and humidity preferences. With this taken into account, I would be curious to determine if the geographic dispersal of Scincidae in Australia appeared to be of any significance in the distribution of the communal nesting behaviour itself, in other words, do populations of skinks that co-habit side by side exhibit communal nesting and isolated populations do not. Another interesting approach would be into the ratio of communal nesting and non-communal nesting within a population. As previously mentioned, communal and solitary nesting often co-exist side by side in one population, but it has not yet been acknowledged which method tends to be preferred in populations, or if individuals have an affinity towards a certain type year after year. These are all engaging and exciting future directions for this type of research. Another fruitful direction of exploration would be to test the many adaptive theories of communal laying such as the predator satiation theory. Identifying the main egg predators of different skink species is of paramount importance in order to pinpoint the ultimate causes of communal laying in reptiles.

Acknowledgments

Special thanks to The University of Newcastle for providing the space and resources required to carry out this investigation, Simon Clulow for generously providing much support and supervision during the experiment. Thanks are extended to Rose Upton, Rhys Corrigan, Lachlan Campbell, and the rest of the team at ‘Frog Lab’ for volunteering to assist where necessary and making our stay very enjoyable.

References

Alfonso, Y.U., Charruau, P., Fajardo, G. and Estrada, A.R. (2012) Interspecific communal oviposition and reproduction of three lizard species in Southeastern Cuba.

Alonso, M., Soberón, R.R., Ramos, R. and Thorbjarnarson, J., 2002, October. Mortality of eggs of Crocodylus acutus associated with the conduct of females in RF Monte Cabaniguán, Cuba. In Poster presented at the 16th Working Meeting of the Crocodile Specialist Group SSS/IUCN (Vol. 710).

Blouin-Demers, G., Weatherhead, P.J. and Row, J.R. (2004) ‘Phenotypic consequences of nest-site selection in black rat snakes ( Elaphe obsoleta )’, Canadian Journal of Zoology, 82(3), pp. 449–456. doi: 10.1139/z04-014.

18

Zoology; Professional Training YearInvestigation into the role of conspecific attraction in communal nesting behaviour of Australian skink sub-species Cryptoblepharus pulcher pulcher1218846Brodie, E.D., Nussbaum, R.A. and Storm, R.M. (1969) ‘An egg-laying aggregation of Five species of Oregon Reptiles’, Herpetologica, 25(3), pp. 223–227. doi: 10.2307/3891399.

Brown, G.P. and Shine, R. (2004) ‘Maternal nest-site choice and offspring fitness in tropical snake (Tropidonophis mairii, Coloubridae)’, Ecology, 85(6), pp. 1627–1634. doi: 10.1890/03-0107.

Brown, G.P. and Shine, R. (2005) ‘Nesting snakes ( Tropidonophis mairii , Colubridae) selectively oviposit in sites that provide evidence of previous successful hatching’, Canadian Journal of Zoology, 83(8), pp. 1134–1137. doi: 10.1139/z05-115.

Cogger, H. (2014) Reptiles and Amphibians of Australia. Available at: https://books.google.co.uk/books?hl=en&lr=&id=q6XfAgAAQBAJ&oi=fnd&pg=PP1&dq=cryptoblepharus+pulcher&ots=VqPNrS6UVX&sig=HblH8tgEx3eiiuVh4Ws7EGqAdUM#v=onepage&q=cryptoblepharus%20pulcher&f=false (Accessed: 16 January 2016).Collias, N.E. and Collias, E.C. (1984) Nest building and bird behavior. United States: Princeton University Press.

Doody, J.S., Freedberg, S. and Keogh, J.S. (2009) ‘Communal egg-laying in Reptiles and Amphibians: Evolutionary patterns and hypotheses’, The Quarterly Review of Biology, 84(3), pp. 229–252. doi: 10.1086/605078.

Ebensperger, L., Hurtado, M.J., Soto-Gamboa, M., Lacey, E. and Chang, A. (2004) ‘Communal nesting and kinship in degus (Octodon degus)’, 91(8). doi: 10.1007/s00114-004-0545-5.

Evans, H. and Hook, A. (1982) ‘Communal Nesting in the Digger Wasp Cerceris australis (Hymenoptera : Sphecidae)’, Australian Journal of Zoology, 30(4), pp. 557–568. doi: 10.1071/ZO9820557.

Graves, B.M. and Duvall, D. (1995) ‘Aggregation of Squamate Reptiles associated with Gestation, Oviposition, and Parturition’, Herpetological Monographs, , pp. 102–119. doi: 10.2307/1466999.

Harris, R.N., Hames, W.W., Knight, I.T., Carreno, C.A. and Vess, T.J. (1995) ‘An experimental analysis of joint nesting in the salamander Hemidaetylium scutatum (Caudata: Plethodontidae): The effects of population density’, Animal Behaviour, 50(5), pp. 1309–1316. doi: 10.1016/0003-3472(95)80046-8.

Horner, J.R. (1982) ‘Evidence of colonial nesting and ’site fidelity’ among ornithischian dinosaurs’, Nature, 297(5868), pp. 675–676. doi: 10.1038/297675a0.Palmer, W.M. and Braswell, A.L. (1976) ‘Communal egg laying and Hatchlings of the rough green snake, Opheodrys aestivus (Linnaeus) (Reptilia, Serpentes, Colubridae)’, Journal of Herpetology, 10(3), pp. 257–259. doi: 10.2307/1562991.

19

Zoology; Professional Training YearInvestigation into the role of conspecific attraction in communal nesting behaviour of Australian skink sub-species Cryptoblepharus pulcher pulcher1218846Noble, Gladwyn Kingsley, and E. R. Mason (1933) Experiments on the brooding habits of the lizards Eumeces and Ophisaurus. American Museum of Natural History, 1933.

Pruett-Jones, S. (1992) ‘Independent versus Nonindependent mate choice: Do females copy each other?’,The American Naturalist, 140(6), pp. 1000–1009. doi: 10.2307/2462930.

Radder, R.S. and Shine, R. (2007) ‘Why do female lizards lay their eggs in communal nests?’, Journal of Animal Ecology, 76(5), pp. 881–887. doi: 10.1111/j.1365-2656.2007.01279.x.

Skutch, A.F. (1961) ‘Helpers among birds’, The Condor, 63(3), pp. 198–226. doi: 10.2307/1365683.

Sternfeld (1918) Search AROD: AROD > Reptiles / Squamata / Scincidae / Cryptoblepharus / elegant snake-eyed skink. Available at: http://www.arod.com.au/arod/reptilia/Squamata/Scincidae/Cryptoblepharus/pulcher (Accessed: 16 January 2016).

Swain, T.A. and Smith, H.M. (1978) ‘Communal nesting in Coluber constrictor in Colorado (Reptilia: Serpentes)’, Herpetologica, 34(2), pp. 175–177. doi: 10.2307/3891671.Swanson, C. (2004) ‘Effect of substrate availability and conspecific cues on communal oviposition in the apple murex snail Phyllonotus pomum’, Marine Ecology Progress Series, 275, pp. 175–184. doi: 10.3354/meps275175.

Tallamy, D.W. (1985) ‘“Egg dumping” In lace bugs (Gargaphia solani, Hemiptera: Tingidae)’, Behavioral Ecology and Sociobiology, 17(4), pp. 357–362. doi: 10.1007/bf00293213.

Wilson, S. and Swan, G. (2003) A complete guide to Reptiles of Australia. Australia: Reed Natural History Australia.

Woolfenden, G.E. (1975) Florida Scrub Jay Helpers at the Nest. Available at: https://sora.unm.edu/sites/default/files/journals/auk/v092n01/p0001-p0015.pdf (Accessed: 29 June 2016).

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Supporting Information

Raw Nest Temperature Data ;

Tub 11/01/2016 (°C) Mean (°C)

Tub 14/01/2016 (°C) Mean (°C)

20


A 25.323.

5 23.4 24 A 25.925.

5 25.9 25.8

B 23.1 22 22.8 22.6 B 26.926.

9 27 26.9

C 24.925.

1 23.3 24.4 2726.

3 25.9 26.4

D 24.525.

5 23.8 24.6 2625.

7 25.3 25.7

E 23.925.

1 23.7 24.2 E 28.428.

1 27.7 28

F 25.625.

1 24 24.9 27.528.

3 28.4 28

G 25.125.

2 25.6 25.3 G 27.327.

5 27.5 27.4

H 25.825.

9 25.8 25.8 H 2827.

9 28.2 28

I 26.1 26 21.9 24.6 I 27.927.

8 27.4 27.7

J 2726.

8 27.3 27 26.427.

1 26.6 26.7Tub 17/01/2016 Mean (°C)

Tub 20/01/2016 Mean (°C)

A 24.623.

7 23.8 24 A 25.125.

1 25.2 25.1

B 24.324.

5 25.1 24.6 B 25.425.

9 26 25.7

25.624.

9 25.2 25.2 C 26.126.

3 26.4 26.3

24.824.

6 25 24.8 D 26.7 27 27.8 27.2

E 25.125.

4 25.4 25.3 E 27.427.

2 27.3 27.3

F 25.725.

9 26.7 26.1 F 27.227.

4 27.5 27.4

G 26.125.

8 25.3 25.7 G 27.227.

1 26.9 27.1

H 25.3 26 26 25.7 H 26.926.

4 26.4 26.6

I 25.725.

5 25.3 25.5 I 26.226.

5 26.5 26.4

2625.

5 25.9 25.8 J 27.1 27 26.8 2726/01/2016 Mean (°C)

Tub 23/01/2016 Mean (°C) A 26 26 26 26B 26 26.5 26.7 26.4C 26.8 27 26.9 26.9D 26.8 27.6 28.5 27.6E 28 27.4 27 27.5F 26.7 27.7 27.6 27.3G 26.3 26.9 26.5 26.6H 25.9 26 26 26I 25.9 26.3 26.7 26.3J 26 26.4 26.7 26.4

21


TubA 25.1 25.5 25.4 25.3B 24.6 25.1 24.7 24.8C 25.1 25.7 25.4 25.4D 26.2 26.6 26.8 26.5E 26.8 26.9 27.4 27F 27.4 27.6 27.6 27.5G 27.3 27.3 27.3 27.3H 27.3 27.9 27.5 27.6I 27.6 27 26.7 27.1J 26.6 27 27.1 26.9

Tub 29/01/2016 Mean (°C) Tub 01/02/2016 Mean (°)A 26.1 26.1 26.2 26.1 A 25.9 26 25.8 25.9B 26.3 26.2 26.3 26.3 B 26.1 25.9 25.3 25.8C 26.4 26.5 26.9 26.6 C 26.9 27 26.6 26.8D 26.9 27.1 27.1 27 D 26.7 26.9 27.2 26.9E 27 27.1 27.3 27.1 E 27.2 27.2 27.4 27.3F 27.3 27.6 27.2 27.4 F 26.9 26.8 27.2 27G 27.2 27.2 27.2 27.2 G 26.7 26.7 27 26.8H 27 26.9 26.8 26.9 H 27.1 27.2 27.2 27.2I 26.5 26.4 26.4 26.4 I 27.4 27.2 27.2 27.3J 27 27.2 27.1 27.1 J 27.5 27.6 27.8 27.6

Tub 04/02/2016 Mean (°C) Tub 07/02/2016 Mean (°C)A 24.6 24.6 24.6 24.6 A 24.9 24.6 24.6 24.7B 25.1 25.1 25.1 25.1 B 24.5 24.5 24.7 24.6C 25.7 25.4 25.3 25.5 C 25.1 25.6 25.4 25.4D 25.8 25.9 25.4 25.7 D 25.4 25.4 25.4 25.4E 25.5 25.7 25.8 25.7 E 24.8 24.8 25.6 25.1F 25.8 25.4 26.4 25.9 F 26.1 26 26 26G 26 25.9 26 26 G 25.7 25.6 25.9 25.7H 25.9 25.4 25.8 25.7 H 25.8 25.7 25.8 25.8I 25.5 25.5 25.6 25.5 I 25.8 25.8 25.9 25.8J 25.5 26.2 25.9 25.9 J 25.8 26.1 26.4 26.1

22


Raw Nest Humidity Data;

Tub 11/01/2016 (%) Mean (%) Tub 14/01/2016 Mean (%)A 90.2 88.1 84.8 87.7 A 83.7 84 85.8 84.5B 84.8 83.6 83.7 84 B 85 83.3 81.3 83.2C 85.7 83.2 86 85 C 86.5 88.1 86.9 87.2D 88.1 88.7 87.8 88.2 D 83.4 85.6 85.3 84.8E 91.8 92.2 91.8 91.9 E 79.8 82.8 82.8 81.8F 86.4 85.9 85.8 86 F 85 84.6 84.6 84.7G 90.5 89.9 90.5 90.3 G 85 86 83.2 84.7H 85.9 86.2 84.7 85.6 H 82.7 83.1 84 83.2

Tub 10/02/2016 Mean (°C) Tub 13/02/2016 Mean (°)A 24.8 24.8 24.9 24.8 A 25.3 25.4 25.4 25.4B 26 25.9 25.7 25.9 B 25.3 25.5 25.8 25.5C 26.1 26.2 26.3 26.2 C 25.6 25.9 26.1 25.9D 26.2 26.1 26 26.1 D 26.2 26.3 26.4 26.3E 25.5 25.9 26.4 25.9 E 26.6 26.9 26.9 26.8F 26.2 25.5 26.6 26.1 F 26.3 27 27.3 26.9G 25.5 25.5 25.6 25.5 G 26.4 26.3 26.5 26.4H 25.7 25.7 25.8 25.7 H 26.4 26.2 26.5 26.4I 25.8 25.6 25.3 25.6 I 25.5 25.8 25.7 25.7J 25.8 26 25.8 25.9 J 26.4 26.9 27.2 26.8

23


I 86.1 85.2 80.6 83.9 I 84.6 85.6 86 85.4J 86.6 88.1 86.7 87.1 J 81.2 82.3 85 82.8

Tub 17/01/2016 Mean (%) Tub 20/01/2016 Mean (%)A 82.6 85.7 85.8 84.7 A 85.1 85.4 86.2 85.6B 84.6 82.8 82 83.1 B 88.2 88.4 88.3 88.3C 84.3 82.7 82.1 83 C 88.5 88.8 89.6 89D 81.9 83.2 82.9 82.7 D 89.4 88.5 89.6 89.2E 83.2 84.7 80.3 82.7 E 87.7 87.2 88.1 87.7F 82.7 84.3 82.1 83 F 87.6 88.2 88.6 88.1G 82.3 75.7 80.3 79.4 G 88.2 89.1 89.3 88.9H 73.8 83.2 81.9 79.6 H 89.8 88.8 89.8 89.5I 84.5 84.4 85.5 84.8 I 89.5 88.9 88.8 89.1

82.8 82.5 82.5 82.6 J 89.7 88.2 87.8 88.6J

Tub 23/01/2016 Mean (%)A 83.8 84.5 84.3 84.2B 84.1 85.3 85.2 84.9C 85.2 85.3 84.5 85D 82.7 77.9 74 78.2E 80.8 82 83.2 82F 85.6 85.5 83.6 84.9G 79 85.4 86.7 83.7H 87.6 88 89.1 88.2I 88.9 89.1 89.7 89.2J 89.6 90.3 90.4 90.1Tub 26/01/2016 Mean (%)

A 86 84.7 84.8 85.2B 85 86.5 84.8 85.4C 85.5 87.6 88 87D 84.3 84.9 83.7 84.3E 84.4 82.2 81.2 82.6F 83.5 80.9 82.5 82.3G 84.2 85.3 84.8 84.8H 83.5 82.9 82.7 83I 83 81.3 85.1 83.1J 85.1 86.6 87.7 86.5

Tub 29/01/2016 Mean (%) Tub 01/02/2016 Mean (%)A 84.1 84.7 84.6 84.6 A 81.6 80.7 81.2 81.2B 85.7 85.4 85.6 85.6 B 79.4 79.4 81 79.9C 85.5 84.6 86 85.4 C 86.3 82.2 80.2 82.9D 84.1 80.1 84.5 82.9 D 81.3 81.6 83.7 82.2E 84.4 84.2 85 84.5 E 82.1 82.4 82.1 82.2F 84.4 85.7 86 85.4 F 84.8 84.3 85.8 85G 85.5 86 86.4 86 G 85.6 85.8 86.7 86H 85.5 85.5 86.5 85.8 H 84.7 84.3 84.1 84.4I 86.2 86.8 87.4 86.9 I 85.2 82.7 82.7 83.5J 88.4 88.8 88.4 88.5 J 82.5 82.4 84.1 83

24


Tub 04/02/2016 Mean (%) Tub 07/02/2016 Mean (%)A 85.8 86.7 86.9 86.5 A 86.5 88 88.9 87.8B 87.3 86.6 86.2 86.7 B 89.5 89.7 90 89.7C 87.4 86.1 85.4 86.3 C 90.3 90.6 90.5 90.5D 86.5 87.6 87.3 87.1 D 91.1 91.4 91.4 91.3E 88.2 88.4 88.5 88.4 E 91.8 91.6 91.7 91.7F 88 88.4 88.1 88.2 F 91.9 91.5 91.9 91.8G 86.5 87.3 87.2 87 G 91.6 91.6 92.1 91.8H 87.4 87.8 87.4 87.5 H 91.9 91.7 91.9 91.8I 87.2 87.1 87.9 87.4 I 91.8 91.5 91.4 91.6J 87.4 88.7 88.8 88.3 J 91.3 91.4 91.4 91.4

Tub 10/02/2016 Mean (%) Tub 13/02/2016 Mean (%)A 84.1 84.6 85.2 84.6 A 85.3 84.9 84.7 85B 83.7 83.5 83.1 83.4 B 85.4 85.5 86.3 85.7C 84.4 83.2 83.1 83.6 C 80.8 85.1 86.9 84.3D 82.3 83.1 84.3 83.2 D 87.1 87.5 86.2 86.9E 82.7 80.9 83.5 82.4 E 84.1 80 80.2 81.4F 86.6 87.7 89.3 87.9 F 86.3 87.6 87.4 87.1G 88.7 89.3 89.5 89.2 G 86.5 88.1 88.3 87.6H 89.3 89 88.9 89.1 H 87.2 87.5 88.3 87.7I 88.4 88.4 88.7 88.5 I 88.7 89.4 89.3 89.1J 89.4 89.8 89.5 89.6 J 82 85.3 80 82.4

Raw Egg Collection Data;

25


Nest

1 2 3 4 5 6 7 8 9 10Tub

Remove Eggs

A27/12 2

29/12 1

31/12 1

B

26/01 28/01 2

26/01 3

C29/01 2

D

E

23/01 02/02 3

Leave Eggs

F

G3/01 2

18/01 2

6/01 18/01 20/01 27/01 7

5/01 26/01 3

20/01 2

H14/01 1

15/01 1

14/01, 17/01 2

26


I27/01 1

6/01 28/01 3

J

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