Nest mass variation over the nesting cycle in the Pied Flycatcher (Ficedula hypoleuca)

The following text is a post-print version of the article: Nest mass variation over the nesting cycle in the Pied Flycatcher (Ficedula hypoleuca) Anna Dubiec and Tomasz D. Mazgajski Avian Biology Research Volume: 6 Issue: 2 Pages: 127 132 DOI: 10.3184/175815513X13612847142708 Published: MAY 2013 The paper has been published in the final form at: http://stl.publisher.ingentaconnect.com/content/stl/abr/2013/00000006/00000002/art00005

Nest mass variation over the nesting cycle in the Pied Flycatcher (Ficedula hypoleuca) Anna Dubiec and Tomasz D. Mazgajski* Museum and Institute of Zoology, Polish Academy of Sciences, Wilcza 64, 00-679 Warszawa, Poland *Corresponding author: mazgaj@miiz.waw.pl ABSTRACT Nest parameters are most commonly measured only once during the nesting cycle. However, the condition of a nest is not constant but can substantially change from one nesting stage to the other, especially between incubation and nestling periods. These temporal changes may drive modifications in nest characteristics. We investigated the change in nest fresh mass between the early stage of incubation and shortly after the nestlings fledged and examined whether nest dry mass measured after the completion of the nesting cycle, which is commonly used in avian studies, is correlated with initial and final nest fresh mass in a small migratory passerine the Pied Flycatcher. Nest fresh mass after the nestlings fledged constituted 180% of the initial fresh mass and the magnitude of mass increase was dependent on the number of fledglings and probably was partly associated with increasing water content in nesting material. Nest dry mass was strongly correlated with nest fresh mass measured both at the beginning of incubation and after the nestlings fledged, and also with the number of fledglings. Because in the Pied Flycatcher changes in the nest fresh mass are dependent on brood characteristics, in order to obtain the most reliable estimate of the initial nest fresh mass, we recommend weighing the nest shortly after its completion, taking care to make the measurement when the humidity of the habitat is rather low. Keywords: Ficedula hypoleuca, fledgling number, nest mass, nest water content 1

1. INTRODUCTION In most bird species nest building is an important component of the nesting cycle. Currently most research interest in this stage of the nesting cycle is driven by exploring the mechanisms behind the considerable variation in nest characteristics at the intra-specific level (e.g. Tomás et al., 2006; Álvarez and Barba, 2008; Mainwaring et al., 2008) as well as estimating the costs of nest construction and its role in shaping life-history trade-offs (Mainwaring and Hartley, 2009; Moreno et al., 2010). In both cases, recording of parameters describing the nest, such as its total size or composition, is essential. In most studies, data on nest characteristics are collected only at a single point during the nesting cycle. However, conditions to which a nest is exposed are not constant, but substantially change from one nesting stage to the other, especially between incubation and nestling period. Consequently, these changes may promote modifications in nest characteristics. For example, nest cup size increases between the incubation and nestling period and in some species may be even 40% wider after than before egg hatching (Slagsvold, 1989) and nests, especially those built of soft material, may become flatter after the start of incubation and later due to nestling activity (Slagsvold, 1989; Lambrechts et al., 2012). Therefore, information on the pattern of such changes may be necessary for proper interpretation of studied phenomena. Mass is the most commonly measured parameter of the nest, which may be used as a reliable estimate of an overall nest size (e.g. Tomás et al., 2006; Mainwaring et al., 2008). It is most frequently measured after the completion of the nesting cycle ( e.g. Alabrudzińska et al., 2003; Mainwaring et al., 2008; Broggi and Senar, 2009). Such timing is probably driven by limitations of earlier measurements associated with possible disturbance of nests with delicate architecture. Generally, only nests that have a rather compact structure, e.g. of Great Tits ( Parus major) and Blue Tits ( Cyanistes caeruleus), may be weighed throughout the whole nesting period during egg laying ( Tomás et al., 2006), incubation (Álvarez and Barba, 2008) or a few days after nestlings hatched (Tomás et al., 2006) without affecting its structure. However, if changes in nest mass over the nesting cycle are not independent of nest/brood characteristics such as the presence of nest-dwelling arthropods or brood size, such estimates may be prone to some bias. Nest fresh mass may be expected to increase with the progress of the nesting cycle as a results of several non-mutually exclusive processes: (1) a continuous supplementation of nesting material, either with the structural material (Vergara et al., 2010), nest lining components such as feathers (McCarty and Secord, 1999) or extra material not considered to 2

be a proper part of the nest (e.g. green plant material, Lambrechts and Dos Santos, 2000), (2) accumulation of various remnants like prey remains and faeces, and 3) changes in humidity/ water content of the nest (Kern and Cowie, 1995). To eliminate variation in nest mass due to the presence of remnants and different levels of humidity/water content, all material not constituting part of the nest is typically removed and the nest is dried at high temperature until a constant mass is reached (e.g. Heeb et al., 2000; Broggi and Senar, 2009; Mainwaring and Hartley, 2009). Despite the strong prerequisites for temporal changes in nest mass, the information on this phenomenon is very scarce (McCarty and Secord, 1999; Tomás et al., 2006; Vergara et al., 2010). In the present study, we examined the variation in nest mass in a small migratory passerine the Pied Flycatcher ( Ficedula hypoleuca) which builds the nest of dry leaves, flakes of bark, moss, roots and grass (Lundberg and Alatalo, 1992). Specifically, we investigated the change in nest fresh mass between the early stage of incubation and shortly after the nestlings fledged, and studied whether this change is associated with brood characteristics (fledgling number) and environmental conditions (rainfall). Moreover, we examined whether nest fresh mass measured at different stages of the nesting cycle is correlated with nest dry mass, which is primarily measured after the nestlings fledge. We suggest that if changes in nest mass over the nesting cycle are consistent and independent of clutch/brood and environmental characteristics, only one measurement taken at any given time of the nesting cycle would be sufficient as a reliable estimate of an overall nest mass. 2. METHODS 2.1. Study area Data were collected in a small (up to 17 pairs per year) nest box breeding population of the Pied Flycatcher located 10 km west of Warsaw (52 05 N, 20 52 E) in a ca. 50-year-old pine forest with no natural breeding sites for hole nesters. The population has been followed from 2005 (except for 2007 and 2008), when the nest box colony, consisting of ca 40 nest boxes was set up. During the breeding seasons of 2011 and 2012, when the data for this study were collected, there were 156 and 188 available nest boxes (floor: 11 11 cm, depth: 21 cm, entrance: 3.2 cm), respectively, positioned in a grid of 50 50 m and ca. 2.5 m above the ground. For the purpose of another project, each year ca 50% of nest boxes was equipped with a wooden insert at the bottom, which decreased the depth of the box by 5 cm. In both study years, all Pied Flycatcher nests were built in boxes with the insert. The front wall of the 3

box is detachable, which allows easy handling of the nest. Nest boxes are primarily occupied by Great Tits and Pied Flycatchers, and occasionally by Blue Tits. After the completion of the breeding season nest boxes were cleaned of remaining nesting material. 2.2. General procedures Starting from the middle of April nest boxes were checked every few days to record laying date (date when the first egg was laid), clutch size, hatching da te, the number of hatchlings and fledglings. When more than one egg was found in the nest, the laying date was backcalculated assuming that one egg was laid per day. In the case of one nest with unknown laying date, time of clutch initiation was calculated based on known hatching date (day = 0) and clutch size and assuming the incubation length of 14 days (Lundberg and Alatalo, 1992). Adult birds and nestlings were ringed with a metal numbered ring, nestlings on day 13 posthatching and adult birds while caught during nestling feeding. In the study area, Pied Flycatcher nests are not very compact, especially during early stages of the nesting cycle, which may cause the disturbance of their structure during the process of extraction from the nest box (own observation, but see Moreno et al. (2009) for the structure of Pied Flycatcher nests in central Spain). To overcome this problem, before each breeding season all nest boxes had a transparent plastic liner (wall height: 5 cm, thickness: 0.2 mm) inserted at the bottom. Such a liner allowed us to remove the nest for weighing and taking measurements without destroying its shape and structure. Similar, but cardboard liners were used by McCarty and Secord (1999) in a study of the Tree Swallow ( Tachycineta bicolor). However, contrary to cardboard, plastic liners do not change the mass due to moisture. In order to allow air circulation, the walls and the bottom of the liner were very densely perforated with a drill (the diameter of the hole 6 mm). Before being inserted into the nest box each liner was weighed to the nearest 0.1 g with a portable electronic balance and later its mass was subtracted from the total mass of the nest and the liner. Nest mass was measured twice during the nesting cycle: shortly after the start of incubation (2 4 days after the clutch completion) and shortly after the nestlings fledged. The liner with the nest was taken out from the box and, after the removal of eggs, weighed to the nearest 0.1 g with an electronic balance. Before the nest was weighed after the nestlings fledged, all materials that were not the part of the nest (dead nestlings, faeces, unhatched eggs, food remnants) were removed. After this measurement nests were transported in plastic bags to the laboratory and placed for at least 48 hours in Tullgren funnels using 60-W electric bulbs as a heat source to extract ectoparasites (data not presented) and weighed again. 4

Because of the exposition to heat, placement of nests in Tullgren funnels may be considered as an equivalent of nest drying at high temperature (until the nest mass remains constant) employed by other studies (e.g. Broggi and Senar, 2009; Mainwaring and Hartley, 2009). Due to a rather low frequency of nest checking (every few days), we cannot exclude that in some cases Pied Flycatcher nests were built on nest material supplied earlier by tits. However, since in most cases tits place only small amounts of nesting material (mostly moss) if they do not later occupy a given box (own observations), this additional material can be neglected while estimating the size of the Pied Flycatcher nest. Two flycatcher nests built on top of tit nests with eggs were excluded from the analyses. Only successful nests, i.e. those in which at least one young fledged, were used in the analyses. Meteorological data from Warszawa-Okecie meteorological station located 9 km from the study site were obtained at http://freemeteo.com. Since Slagsvold (1989) demonstrated that water content of Pied Flycatcher nests decreases rather rapidly and nests with water content of over 70% may almost completely dry out (at room temperature with air humidity of ca 30%) within 3 days, we assessed whether changes in nest fresh mass may be associated with the humidity of the surrounding habitat reflected by the total rainfall within 3 days preceding the measurements. 2.3. Statistical analyses Two females nested during both breeding seasons, each time with a different male. To meet the assumption of independent sampling, only one breeding attempt was selected for the analyses. The effect of year on clutch characteristics, nest fresh and dry mass and total rainfall within 3 days preceding the measurement of the nest fresh mass was tested with t test or Mann-Whitney U test (in case of variables, in which transformations failed to normalise its distribution or the assumption of equality of variances was violated). The effect of humidity in the surrounding habitat on the change in nest fresh mass was tested with ANOVA with a two-level factor describing humidity: total rainfall during 3 days preceding the measurement equalling 0 and above 0. The associations between nest mass/changes in nest mass and other variables were tested with Pearson s correlation coefficient. Sample sizes differ between the analyses, because not all data were collected for each nesting attempt. Three nests, in which one egg was broken during handling, were included in the analyses since hatching success was not in the focus of this study. The analyses were performed in Statistica ver. 7.1 (StatSoft 2005). 5

3. RESULTS In 2011, Pied Flycatchers initiated the clutch on average 6 days later than in 2012, however, neither clutch size, the number of fledglings, nest mass nor total rainfall during 3 days preceding the nest fresh mass measurements differed between the breeding seasons (Table 1). Since we considered that the composition of nesting material and hence the properties of the nest would be similar in both years, and we focused on within-nest changes in mass, which should be dependent on specific weather conditions preceding the nest measurements rather than some general year-related effects, we pooled data from two breeding seasons. During early incubation nests weighed on average 26.81 ± 7.83 (SD) g (n = 21, range: 17.0 45.3 g) and shortly after the nestlings fledged 47.62 ± 12.77 g (n = 21, range: 29.9 74.3 g). On average nest fresh mass increased between these two measurements by 20.80 ± 10.98 g (n = 21, range: 5.1 49.4). The magnitude of this change was not affected by rainfall within 3 days preceding the second measurement (F 1, 19 = 1.779, P = 0.198). However, nests which fledged more nestlings gained more mass between these two measurements (r = 0.72, n = 20, P < 0.001, Figure 1), which could be at least partly attributed to accumulation of larger amounts of water (final nest fresh mass nest dry mass) in nests with large than with small broods (r = 0.54, n = 17, P = 0.024). Moreover, although the fledgling number was not associated with initial nest fresh mass (r = 0.13, n = 20, P = 0.594), it positively correlated with nest dry mass (r = 0.72, n = 17, P = 0.001). In response to drying in Tullgren funnels, nest material lost on average 8.82 ± 4.79 g (n = 18, range: 3.5 23.8) and weighed 39.43 ± 10.67 g (n = 18, range: 25.1 63.2). Nest dry mass was very strongly correlated with fresh mass measured after the nestlings fledged (r = 0.95, n = 18, P < 0.001) and strongly with fresh mass at the beginning of incubation (r = 0.76, n = 18, P < 0.001; Figure 2). The relation between dry and fresh mass was also assessed separately in groups of nests in which the measurement of fresh mass was preceded or not by rainfall. In the case of the fresh mass measured at early incubation, the correlation with nest dry mass was weaker when measurement was preceded by some rainfall (rainfall: r = 0.70, n = 12, P = 0.011, no rainfall: r = 0.92, n = 6, P = 0.010). Similar trend was observed for correlation between dry mass and fresh mass measured after the nestlings fledged (rainfall: r = 0.94, n = 13, P < 0.001, no rainfall: r = 0.99, n = 5, P < 0.001). 6

4. DISCUSSION Surprisingly few studies have looked so deeply into the pattern of changes in nest mass over the nesting cycle in birds. Scarcity of such data may be at least partly associated with a delicate nest architecture in many species, which makes it difficult to extract the nest without disturbing its structure. Use of a plastic liner allowed us to show that nest fresh mass after the nestlings fledged constituted 180% of the initial nest fresh mass. Interestingly, the magnitude of nest fresh mass increase was associated with the number of nestlings, which fledged from a given nest. Specifically, the more nestlings fledged, the more nest mass increased which could, at least partly, be attributed to higher amounts of water retained in nests with large broods. Moreover, nest dry mass measured after the completion of the nesting cycle was a very strong predictor of fresh mass after the completion of the nesting cycle and a strong predictor of fresh mass during incubation. Tomás et al. (2006) showed in the Blue Tit that nest fresh masses weighed during egg laying and on day 3 post-hatching were very strongly correlated, however, the authors did not provide data on the difference in mean mass between these two measurements and based the correlation on a very small sample size. McCarty and Secord (1999) found an increase in nest fresh mass with the progress of the nesting cycle in the Tree Swallow. The magnitude of this increase was rather low, which may be attributed to both relatively short time between two measurements (from the day when the first egg was laid until the day of egg hatching) and not including the nestling period, when changes in nest mass are probably most distinct due to the activity of nestlings. Since Tree Swallows add feathers to the nest until the early stages of the nestling period ( Lombardo, 1994), the increase in the total nest fresh mass may be at least partly attributed to new material deposited in the nest. In the Pied Flycatcher nest construction is completed before the onset of egg laying with little amounts of nesting material sometimes added on the day the first egg is laid (Stjernberg, 1974). Therefore, such process may not contribute to the observed changes in nest mass. In the Pied Flycatcher increase of the nest fresh mass over the nesting cycle is probably primarily associated with accumulation of water and dust in nesting material. Generally, water content in the nest may be affected by both abiotic (e.g. prevailing weather conditions) and biotic factors (e.g. nestlings and nest-dwelling arthropods). Since Pied Flycatcher nests in the study area contain only scarce amounts of mosses which are highly water absorbent, nests of this species are not expected to very closely reflect the humidity of the habitat (Eeva et al., 1994). In accordance with this assumption, water content in the nest was not associated with 7

total rainfall within a few days preceding the measurement. Nest water content in this species seems to be rather affected by biotic factors, specifically nestlings. We found that nests containing more nestlings accumulated more water. Such relation may arise if total metabolic turnover increases with brood size which, in turn, should result in higher evapotranspiration, possibly affecting the nest humidity (Heeb et al., 2000). Additionally, nests with large broods may retain more water extracted from food remnants and faeces, which quantity should depend on the number of nestlings. Brood size has been also found to affect nest humidity (measured as the total mass of water in the nest divided by nest dry mass) in the Great Tit (Heeb et al., 2000). Alternatively or in combination with brood size, nest water content may be affected by the presence of nest-dwelling arthropods, especially ectoparasitic ones. For example, in Great and Blue Tits high infestation with Protocalliphora blow fly larvae is often associated with wetter nests than usual (Heeb et al., 2000; Mennerat et al., 2009) and in the Great Tit nest humidity has been also found to be much higher in flea-infested than uninfested nests (Heeb et al., 2000). Consequently, if nests with large broods are inhabited by large number of ectoparasites, as has been shown for Great Tits (Eeva et al., 1994, but see Heeb et al., 1996), this could explain their higher water content. However, this mechanism may be much less important in the Pied Flycatcher than tit nests for two reasons. Firstly, in this species nest water content seems to be not associated with the infestation level with fleas and Protocalliphora larvae (Eeva et al., 1994) and secondly, the load of ectoparasites is independent of brood size (Eeva et al., 1994). In case of dust-associated changes in nest mass, they may be primarily linked with dust produced in the process of feather growth. In Great Tits and Blue Tits dust is the second heaviest component of the nest when measured after the completion of the nesting cycle and its amounts are positively correlated with the number of fledglings (Britt and Deeming, 2011). Since in this study mass of different nest components was not measured, we may not directly verify whether in the Pied Flycatcher dust contributed to temporal increase in nest mass. However, the positive correlation between the number of fledglings and nest dry mass, with a simultaneous lack of such association with initial fresh mass, may indicate that temporal changes in nest mass, which are unrelated to water content, are affected by the number of fledglings. Nest dry mass has been shown to be very strongly correlated with fresh mass after the completion of the nesting cycle. However, it was a less reliable, although still strong, predictor of the initial fresh mass. The humidity of the surrounding habitat seemed to affect 8

this association since the correlation was stronger when the fresh mass was measured following a few days without rain. 5. CONCLUSIONS In the Pied Flycatcher nest, fresh mass considerably increases with the progress of the nesting cycle. Importantly, the magnitude of this change is dependent on the number of nestlings leaving the nest. Nest dry mass is a strong predictor of nest fresh mass measured shortly after the completion of nest construction and after the nestlings fledged. However, since the changes in nest mass in this species are dependent on brood characteristics, specifically brood size, in order to obtain the most reliable estimate of the initial nest mass we recommend weighing the nest shortly after its completion, taking care to take the measurement when the humidity of the habitat is rather low. ACKNOWLEDGEMENTS We thank Iga Góźdź and Bartosz Matuszczak for help in the field. The study was financially supported by grant no. N304 345139 from the Polish Ministry of Science and Higher Education/National Science Centre. REFERENCES Alabrudzińska, J., Kaliński, A., Słomczyński, A., Wawrzyniak, A., Zieliński, P. and Bańbura, J. (2003) Effects of nest characteristic o n breeding success of Great Tits Parus major. Acta Ornithol., 38, 151 154. Álvarez, E. and Barba, E. (2008) Nest quality in relation to adult bird condition and its impact on reproduction in Great Tits Parus major. Acta Ornithol., 43, 3 9. Britt, J. and Deeming, D.C. (2011) First-egg date and air temperature affect nest construction in Blue Tits Cyanistes caeruleus, but not in Great Tits Parus major. Bird Study, 58, 78 89. Broggi, J. and Senar, J.C. (2009) Brighter Great Tit parents build bigger nests. Ibis, 151, 588 591. 9

Eeva, T. Lehikoinen, E. and Nurmi J. (1994) Effects of ectoparasites on breeding success of Great Tits ( Parus major) and Pied Flycatchers ( Ficedula hypoleuca) in an air pollution gradient. Can. J. Zool., 72, 624 635. Heeb, P., Kölliker, M. and Richner, H. (2000) Bird -ectoparasite interactions, nest humidity and ectoparasite community structure. Ecology, 81, 958 968. Heeb, P., Werner, I., Richner, H. and Kölliker, M. (1996) Horizontal transmission and reproductive rates of hen fleas in Great Tit nests. J. Anim. Ecol., 65, 474 484. Kern, M.D. and Cowie, R.J. (1995) Humidity levels in Pied Flycatcher nests measured using capsule hygrometers. Auk, 112, 564 570. Lambrechts, M.M. and Dos Santos, A. (2000) Aromatic herbs in Corsican Blue Tit nests: the Potpourri hypothesis. Acta Oecol., 21, 175 178. Lambrechts, M.M., Aime, C., Midamegbe, A., Galan, M.J., Perret, P., Gregoire, A. and Doutrelant, C. (2012) Nest size and breeding success in first and replacement clutches: an experimental study in Blue Tits Cyanistes caeruleus. J. Ornithol., 153, 173 179. Lombardo, M.P. (1994) Nest architecture and reproductive performance in tree swallows (Tachycineta bicolor). Auk, 111, 814 824. Lundberg, A. and Alatalo, R.V. (1992) The Pied Flycatcher. T & A.D. Poyser, London. Mainwaring, M.C., Benskin, C.McW.H. and Hartley, I.R. (2008) The weight of female-built nests correlates with female but not male quality in the Blue Tit Cyanistes caeruleus. Acta Ornithol., 43, 43 48. Mainwaring, M.C. and Hartley, I. (2009) Experimental evidence for state -dependent nest weight in the Blue Tit, Cyanistes caeruleus. Behav. Proc., 81, 144 146. McCarty, J.P. and Secord, A.L. (1999) Nest -building behavior in PCB-contaminated tree swallows. Auk, 116, 55 63. Mennerat, A., Mirleau, P., Blondel, J., Perret, P., Lambrechts, M.M. and Heeb, P. (2009) Aromatic plants in nests of the Blue Tit Cyanistes caeruleus protect chicks from bacteria. Oecologia, 161, 849 855. Moreno, J., Merino, S., Lobato, E., Ruiz-De-Castañeda, R., Martínez-De la Puente, J., Del Cerro, S. and Rivero-De Aguilar, J. (2009) Nest-dwelling ectoparasites of two sympatric hole-nesting passerines in relation to nest composition: An experimental study. Ecoscience, 16, 418 427. Moreno, J. Lobato, E., González-Braojos, S. and Ruiz-de Castañeda, R. (2010) Nest construction costs affect nestling growth: a field experiment in a cavity-nesting passerine. Acta Ornithol., 45, 139 145. 10

Slagsvold, T. (1989) Experiments on clutch size and nest size in passerine birds. Oecologia, 80, 297 302. StatSoft (2005) STATISTICA (data analysis software system), version 7.1. http://www.statsoft.com Stjernberg, M. (1974) Nest -building by the Pied Flycatcher Ficedula hypoleuca. Ornis Fennica, 51, 85 109. Tomás, G., Merino, S., Moreno, J., Sanz, J.J., Morales, J. and Garcia-Fraile, S. (2006) Nest weight and female health in the Blue Tit (Cyanistes caeruleus). Auk, 123, 1013 1021. Vergara, P., Gordo, O. and Aquirre, J.I. (2010) Nest size, nest building behaviour and breeding success in a species with nest reuse: the white stork Ciconia ciconia. Ann. Zool. Fennici, 47, 184 194. 11

Table 1. Between-year comparison (mean ± SD) of clutch characteristics, nest fresh and dry mass and total rainfall within 3 days preceding the measurement of the nest fresh mass in the Pied Flycatcher population from central Poland. Differences were tested either with t test or Mann-Whitney U test; n denotes sample size. 2011 n 2012 n Statistics Laying date (day 1 = 1 May) 15.4 ± 2.4 10 9.6 ± 4.3 11 t 19 = 3.72, P = 0.001 Clutch size 7.0 ± 0.8 10 6.5 ± 0.7 11 z = 1.23, P = 0.218 Fledgling number 5.7 ± 1.16 10 5.6 ± 1.35 10 t 18 = 0.18, P = 0.861 Incubation nest fresh mass (g) 28.9 ± 8.0 10 25.0 ± 7.5 11 t 19 = 1.15, P = 0.264 Post-fledging nest fresh mass (g) 48.0 ± 13.5 10 47.3 ± 12.8 11 t 19 = 0.13, P = 0.895 Nest dry mass (g) 41.6 ± 13.3 7 38.1 ± 9.0 11 t 16 = 0.68, P = 0.506 Total rainfall (mm) (incubation) 3.8 ± 4.0 10 10.4 ± 10.2 11 z = -1.23, P = 0.218 Total rainfall (mm) (post-fledging) 6.7 ± 3.9 10 7.0 ± 7.9 11 z = -0.14, P = 0.888 12

Figure 1. Increase in the nest fresh mass between the early incubation period and after the nestlings fledged in relation to the number of fledglings in the Pied Flycatcher. 60 Increase in nest fresh mass (g) 50 40 30 20 10 0 4 5 6 7 8 Fledgling number Figure 2. Relation between post-fledging nest dry mass (measured after at least 48 hours in Tullgren funnels) and nest fresh mass measured at two points of the nesting cycle at the beginning of the incubation period and after the nestlings fledged in the Pied Flycatcher. 80 70 mass measured at incubation mass measured post-fledging 60 Nest fresh mass (g) 50 40 30 20 10 10 20 30 40 50 60 70 80 Nest dry mass (g) 13