Parasite lineages vary widely in species richness. In some clades, speciation is linked to the colonisation of new hosts. This is the case in the indigobirds and whydahs (Vidua), brood-parasitic finches whose nestlings mimic the phenotypes of their specific hosts. To understand the factors limiting host colonisation, and therefore speciation, we simulated the colonisation of a host using cross-fostering experiments in the field. Despite DNA barcoding suggesting that host species feed their chicks similar diets, nestling Vidua had low survival in their new host environment. Nestling Vidua did not alter their begging calls plastically to match those of the new hosts, and were fed less compared to both host chicks and to Vidua chicks in their natural host nests. This suggests that a key hurdle in colonising new hosts is obtaining the right amount rather than the right type of food from host parents. This highlights the importance of mimetic nestling phenotypes in soliciting feeding from foster parents and may explain why successful colonisations tend to be of hosts closely-related to the ancestral one. That non-mimetic chicks are fed less but not actively rejected by host parents suggests how selection from hosts can be sufficiently intense to cause parasite adaptation, yet sufficiently relaxed that parasitic chicks can sometimes survive in and colonise new host environments even if they lack accurate mimetic phenotypes. The difficulties of soliciting sufficient food from novel foster parents, together with habitat filters, likely limit the colonisation of new hosts, and therefore speciation, in this parasite radiation.

Transfer experiments simulating host colonisation

During January–April 2014–2017, we carried out transfer experiments within an area of about 40 km² on and around Musumanene and Semahwa Farms (centred on 16°47′S, 26°54′E) in the Choma District of Zambia. The habitat is a mixture of miombo woodland, agricultural fields and seasonally-flooded grasslands. 

 

The experiment had three treatments: (i) pin-tailed whydah eggs transferred from common waxbill to blue waxbill nests, (ii) blue waxbill eggs transferred to blue waxbill nests, and (iii) common waxbill eggs transferred to blue waxbill nests. Additionally, we monitored survival and feeding of pin-tailed whydah nestlings in naturally-parasitised common waxbill nests. To minimise predation, eggs were taken from their natural nest and incubated in a Brinsea Octagon 20 Advance EX Incubator at 36.7°C and 60% humidity. A day before they were due to hatch, eggs were fostered to a blue waxbill nest. Occasionally (16/94 transfers), the offspring had to be fostered as a chick freshly hatched in the incubator, rather than as an egg but this was not found to influence the offspring’s subsequent survival in the novel nest environment (see Results). The modal clutch size of both common waxbill and blue waxbill nests was five. No host egg was removed when an egg/hatchling was added, because pin-tailed whydah females do not remove a host egg when they naturally parasitise a nest (Tarboton, 2011).  Experimental nests were visited every two or three days, and the number of eggs and chicks in the nest was recorded. For chicks, mass and tarsus length were measured and the amount of food in the crop scored (methods below). Mass was measured on digital scales to an accuracy of 0.1 or 0.01 g depending on the model of scale. Tarsus length was measured using dial callipers to the nearest 0.1 mm.

 

All three species used in the study are common and widespread and experience high levels of natural nest predation such that the experiments carried out for this study will have negligible effects on their populations. Data were collected under the research approval of the Department of National Parks and Wildlife in Zambia (DNPW/8/27/1). Our sample sizes were chosen to allow the between-species effects to be detected with a high degree of confidence while not including an unnecessarily high number of individuals. As such it meets the ABS/ASAB guidelines and adheres to the three R’s of replacement, reduction and refinement (Buchanan et al., 2012).

Comparing survival of different species transferred to blue waxbill nests

Survival analyses were carried out in the R statistical environment(R Development Core Team, 2017) using the packages Survival (Therneau, 2015) and KMsurv (Moeschberger and Yan, 2012). We monitored chick survival from the day the chick hatched in the new host nest. Chick survival was judged to end at the midpoint between the last day the chick was known to be alive and the first day the chick was known to be absent. If the nest was still active, but the transferred chick was absent, then the chick was assumed to have died. If the nest was abandoned at the point the transferred chick was absent, then the data were right-censored. Right-censoring is used when the event of interest has not occurred by the last observation (Mills, 2011). A Cox proportional hazards model was fitted to the survival data (Cox, 1972). The co-variates in the initial model were: (i) transferred chick species, (ii) presence of host nestmates, and (iii) foreign chick transferred as egg or as chick. The number of nestmates over the course of a given transfer experiment ranged from 0 to 5 (mean = 1.4). In most transfers (49 of 94), the transferred chick had no nestmates in the foster nest. We therefore modelled nestmate presence as presence/absence.

 

Comparing the amount of food host parents fed transferred chicks of different species

Crop size of the transferred chick was recorded at each nest visit. Nestling estrildid crops are transparent, allowing easy visual inspection. Crops were scored as 0 (empty), 1 (trace amounts, < c. 20 seeds, no bulge in crop), 2 (> c. 20 seeds, slight bulge) or 3 (> c. 50 seeds, large bulge). To assess whether crop sizes of chicks differed depending on the species of chick transferred, two approaches were used: 

 

First, the median crop size of the transferred chick over the first 7 days of survival in the host nest was used as the response variable; c. 80% of common waxbill and pin-tailed whydah chick mortality occurred in this period (Figure 2). A Kruskal-Wallis test was used to test whether median crop size differed between the three species. Median crop sizes were compared between species using a Dunn post-hoc test, using the dunn.test function from the R package dunn.test and with Bonferroni correction for multiple testing (Dinno, 2017). 

 

Second, ordinal mixed-effect models were implemented in the R package Ordinal (Christensen, 2015) using crop score as an ordinal response variable. In the full model, the fixed effects were chick species and the number of nestmates. Transferred chick individual was a random effect nested within the nest of origin of that transferred chick. We carried out stepwise elimination of non-significant co-variates until only significant co-variates remained. The model was initially run to include crop scores over the first 7 days of development, then re-run using crop scores over the first 4, 5, 6 and 8 days of development to test whether the findings from the first 7 days of development were robust. 

 

Ordinal mixed-effect models were used to compare crop size in pin-tailed whydahs and common waxbills occurring in their natural nests (common waxbill nests) with those experimentally transferred to blue waxbill nests. Data for pin-tailed whydahs and common waxbills in common waxbill nests are observational, unlike the experimental data from pin-tailed whydahs in blue waxbill nests. This was because high levels of nest predation meant that all common waxbill nests found at the egg stage were required as a source of eggs for transfer to blue waxbill nests. Therefore, our data on pin-tailed whydahs in common waxbill nests do not account for the effects of transferring an egg from one nest to another. However, when blue waxbill eggs were transferred from their own nest to another blue waxbill nest, they still showed high survival (about 76% of all chicks transferred survived to fledging), suggesting that any effects of transferring eggs between nests are insufficient to account for the differences observed in chick survival between common and blue waxbill nests.

 

Measuring nestling diet composition

Obtaining crop samples in field

Nestling crops were sampled using the tube insertion method (Zann and Straw, 1983). The tube was inserted in the throat of the nestling and seeds were pushed from the translucent crop into the tube. The contents were stored in 70% ethanol. The process was repeated until about 20–30 seeds had been extracted. Chicks were sampled around the time when the primaries first erupt from pin (approximately 6–7 days of age).

 

We sampled crops of common waxbill, blue waxbill as well crops of six other sympatric estrildid finch species: orange-winged pytilia (Pytilia afra), melba finch (Pytilia melba), Jameson’s firefinch (Lagonosticta rhodopareia), red-billed firefinch (L. senegala), African quailfinch (Ortygospiza atricollis) and bronze mannikin (Spermestes cucullatus). We sampled these other estrildid finch species in addition to the two used in the experiments to assess variation in host diet across a broader phylogenetic scale and to explore whether estrildids that hosts to Vidua have a different diet from those that are not.

 

DNA barcoding of nestling crop contents

Nestling estrildid finch crops contained almost exclusively plant seeds. DNA barcoding of samples was carried out by Jonah Ventures (Boulder, Colorado; jonahventures.com). The chloroplast trnL intron was amplified from DNA in each sample using the c and h trnL primers (Taberlet et al., 2007). The total expected amplicon length was 332bp (Jonah Ventures in litt.). A detailed protocol is described in supplementary methods. We consulted with an expert botanist based in Zambia, Mike Bingham, to validate the taxonomic identities assigned by DNA barcoding.

 

Quantifying crop contents

DNA barcoding data resolution allowed analysis at the subfamily level and not the genus level, so each OTU was assigned to one of the four subfamilies identified (see Results). For each sample, reads from OTUs mapping to the same sub-family were summed together to give a measure of the total number of reads from each subfamily, and expressed as a proportion of total reads (Craine et al., 2015; Willerslev et al., 2014). To test whether different estrildid species fed chicks different proportions of seeds from each of the four families, non-metric dimensional scaling (NMDS) was performed using the R package vegan (Oksanen et al., 2017). Comparisons of diet between species were made using the function adonis, from the R package vegan.

 

Begging call plasticity

Recording nestling begging calls

Chicks were placed in an artificial nest, and given several minutes for acclimation. To stimulate begging, the chick was tapped gently with forceps on the bill. Recordings were made using an Audio-Technica ATR35S tie-clip microphone or a Sennheiser ME-66 shotgun microphone held approximately three cm away from the chick’s mouth. Recordings were made for around two minutes or until at least ten seconds of continuous begging were recorded.

 

Analysing the effect of host environment on nestling begging calls

We compared begging calls of nestling pin-tailed whydahs in natural common waxbill nests, to those transferred to blue waxbill nests. We identified four distinct call types both by listening to recordings and through visual inspection of sonograms (see Results). All four call types were detected in both pin-tailed whydahs developing in common waxbill nests, and pin-tailed whydahs transferred to blue waxbill nests. To analyse whether host environment influenced the stage in the nestling period at which each call type was made, we examined the stage in development at which chicks made each call type and compared this between pin-tailed whydahs raised in common and blue waxbill nests. We used chick tarsus length as a proxy for developmental stage, because for pin-tailed whydahs in their natural nests, the exact age in days of the chick was unknown, whereas tarsus length was available for all treatments. 

 

We examined whether within each call type, there were changes in call structure between host environments. For each call type, the following begging call parameters were extracted from each recording: minimum frequency, maximum frequency, centre frequency, peak frequency, frequency bandwidth, call duration, average entropy, and energy. These are widely used in the literature to characterise begging sounds (Anderson et al., 2009; Butchart et al., 2003; Langmore et al., 2008). For each recording, ten sequential call notes in a bout of begging were selected and call parameters extracted. Call notes were not selected if they overlapped with interfering background noises, or if they were incomplete calls. The relationship between the call types was visualised using linear discriminant function analysis with the R package MASS (Venables and Ripley, 2002). We calculated call rate by dividing the dividing the number of calls in the bout by the duration of the bout.

 

Two approaches were used to compare the structure of each call type between pin-tailed whydahs raised in common waxbill nests, and those raised in blue waxbill nests. First, a series of linear mixed models were constructed, with each call parameter as a separate response variable. Host environment and crop size were fitted as fixed effects and individual chick identity as a random effect. Crop size was used as a proxy for chick hunger. When estrildid finch nestlings are fed, they store their seed in the crop before passing it on to the stomach. By measuring the amount of food stored in the crop, we could assess how much the chick had recently been fed in a non-invasive manner. Crops were scored as 0 (empty), 1 (trace amounts, < c. 20 seeds, and with no bulge in crop), 2 (> c. 20 seeds and with slight bulge) or 3 (> c. 50 seeds and with large bulge). We controlled for multiple testing using Bonferroni correction (Dunn, 1961). Second, we carried out a logistic regression analysis using the R package nnet (Venables and Ripley, 2002),  allowing all call parameters to be considered at once.