DOI: 10.5061/dryad.dbrv15f8g
title: Multi-dimensional niche differentiation of two sympatric breeding secondary cave-nesting birds in Northeast China using DNA metabarcoding

description: Niche theory predicts that ecologically similar sympatric species should
 show differentiation in at least one of the main niche dimensions (time,
 space, and/or food). Here, we combined observations of breeding timing,
 nest site selection, and diet (the latter determined using DNA
 metabarcoding) to analyze the niche overlap and differentiation between
 two sympatric secondary cavity-nesting birds, the Japanese Tit Parus minor
 and the Yellow-rumped Flycatcher Ficedula zanthopygia. The results showed
 that 1) there were significant differences in the first egg laying date,
 length of the egg laying period, incubation date, and hatching date
 between tits and flycatchers, and the breeding time of flycatchers peaked
 later (about 30 days) than that of tits; 2) the two species had a large
 overlap in nest site selection, although the canopy coverage and shrub
 density of flycatchers were significantly higher than those of tits; and
 3) the niche overlap in diet was minimal, with both species heavily
 relying on Lepidoptera (39.6% and 63.7% for tits and flycatchers,
 respectively), but with flycatchers consuming significantly higher
 percentages of Lepidoptera, Diptera, and Coleoptera than tits. The results
 indicate that these two sympatric secondary cavity-nesting species have
 significant niche differentiation in breeding time and diet, but little
 differentiation in nest site selection.
description: <strong>Study area</strong> The
 study was conducted during two breeding seasons (2021–2022) in Zuojia
 Nature Reserve, Jilin, northeastern China (44°1ʹ–45°0ʹN, 126°0ʹ–126°8ʹE).
 The reserve has an area of 60 km². The altitude is 200–554.6 m, with a
 continental monsoon climate in the temperate zone. The average annual
 precipitation is 680 mm, and the average temperature is 5–6°C. The
 vegetation is a natural secondary broad-leaved forest (Deng<em>
 </em>et al., 2011), including <em>Betula dahurica</em>,
 <em>Fraxinus mandschurica</em>, <em>Quercus
 mongolicus</em>, and <em>Tilia mandshurica</em>.
 Artificial nesting boxes (12 cm × 12 cm × 25 cm<sup>
 </sup>inside, 4.5 cm diameter of the hole) were hung in the study
 area to attract the tits and flycatchers. The nesting boxes were randomly
 attached to trees at a height of about 2.5–3 m above the ground, with a
 minimum distance of 30 m between each box. The tree species and nesting
 orientation were assigned at random, and the number of nesting boxes was
 maintained at about 450 year round (Yu et al., 2017).
 <strong>Data collection</strong>
 <strong>Collection of breeding time
 data</strong> We visited the nest boxes every 3
 days after mid-March to monitor the reproductive activities and clutch
 sizes of these two species (Fan<em> </em>et al., 2021). We
 recorded the first egg-laying date, incubation date, and hatching date of
 the tits and flycatchers. The breeding time did not include the nestling
 period in this study due to the difficulty of accurately monitoring the
 fledging date of each nest. Breeding time data were continuously recorded
 for 152 and 26 nests of tits and flycatchers, respectively. In addition,
 to measure reproductive fitness, we recorded the brood size of both
 species and calculated the hatching rate (i.e., the ratio of brood size to
 clutch size). We collected hatching data from 57 nests of tits and 26
 nests of flycatchers between mid-May and late June, when both species were
 breeding and experiencing interspecific competition.
 <strong>Collection of nest site
 data</strong> We measured 13 nest site
 characteristics (Li et al., 2023) of the nest boxes occupied by tits and
 flycatchers. The nest height (m) (NH) above ground, the nest tree diameter
 (cm) at breast height (DBH), and the average diameter (cm) at breast
 height (ADBH) of 10 trees within a 10 m radius circle centered on the
 nesting tree were measured using a tape measure. The nest tree height (m)
 (TH), average height (m) of 10 trees (ATH) within a 10 m radius circle
 centered on the nesting tree, the average height (cm) of 10 shrubs (ASH),
 and the shrub density (SD) within a 1 m radius circle centered on the
 nesting tree were measured using a laser rangefinder. We recorded the nest
 tree species (TS), the number of tree species (NTS), and the number of
 trees (NT) within a 10 m radius circle centered on the nesting tree and
 assessed canopy cover (%) (CC) above the nest tree. The CC was measured by
 standing 1 m away from the nest tree in each of the four cardinal
 directions and observing it vertically to assess it (Li et al., 2023). The
 entrance inclination (EI) of nest boxes was measured by a digital
 protractor (Weidu Electronics Co., LTD, Wenzhou, China). The orientation
 of the nest box entrance (OE) was measured by a compass. We collected data
 for 300 nests of tits and 34 nests of flycatchers. The nest site data
 included nests with complete breeding time data, as well as those with
 incomplete data (i.e., missing hatching dates or nests preyed upon by
 predators). <strong>Collection of adult
 feces</strong> From mid-May to early June, adults
 of tits and flycatchers breeding in artificial nest boxes were captured
 using the baffle method after the nestlings had hatched (Fan et al.,
 2021). The adults were placed in a cage (18 cm × 12 cm × 8 cm) lined with
 sterile craft paper by an experimenter wearing sterile latex gloves. These
 individuals usually defecated naturally within 5 min, and the feces were
 collected using sterile polyester swabs and placed in lyophilizing tubes
 (DNase/RNase-Free, Sterile) containing RNAlater (Servicebio, Wuhan,
 China). The lyophilizing tubes were gently inverted to completely immerse
 the feces in the RNAlater. The birds were released in place after they
 were ring banded. We obtained 49 fecal samples from adult tits across 32
 nest boxes and 24 fecal samples from adult flycatchers across 19 nest
 boxes. The tubes containing feces were temporarily placed in a thermal bag
 with ice packs and then stored at −80°C until DNA extraction.
 <strong>DNA extraction, PCR, and
 sequencing</strong> Total DNA from collected
 fecal samples was extracted using a PowerSoil® DNA Isolation Kit (MoBio
 Laboratories, Carlsbad, CA, USA) according to the manufacturer's
 instructions. After DNA extraction, DNA integrity was tested using 1%
 agarose gels. We used the primers LCO-1490 (GGTCAACAAATCATAAAGATATTGG) and
 ZBJ-ArtR2c (WACTAATCAATTWCCAAATCCTCC) to amplify a 225-bp fragment of the
 cytochrome c oxidase subunit I (COI) barcode region (Folmer et al., 1994;
 Zeale et al., 2011). Sample-specific 8-bp barcodes were attached to the
 5' ends of the primers. The PCR amplification was conducted in a
 final volume of 20 μl containing 4 μl of 5 × FastPfu Buffer, 2 μl of dNTPs
 (2.5 mM), 0.8 μl of primer F (5 μM), 0.8 μl of primer R (5 μM), 0.2 μL of
 Bovine Serum Albumin (BSA), 0.4 μl of FastPfu Polymerase, and 10 ng of
 DNA. The conditions for PCR were as follows: an initial denaturation step
 at 95°C for 3 min; 95°C for 30 s, 52°C for 30 s, and 72°C for 30 s, for a
 total of 45 cycles, followed by a final extension at 72°C for 10 min. Each
 PCR batch included 1–2 PCR blanks. Three separate PCR replicates were
 conducted for each extract, and these were then mixed after PCR
 amplification. After amplification, the PCR products were visualized by
 electrophoresis using 1% agarose gels and purified with Agencourt AMPure
 XP (Beckman Coulter, Indianapolis, IN, USA). The purified products were
 paired-end sequenced on an Illumina Miseq PE300 platform (Illumina Inc.,
 SanDiego, CA, USA) by Beijing Ovison Gene Technology Co., Ltd (Beijing,
 China). <strong>Sequencing data
 analysis</strong> Raw sequences were split using
 QIIME v1.8.0 (Caporaso<em> </em>et al., 2010) based on the
 barcode tags. Paired-end reads were merged and quality-filtered by Pear
 v0.9.6 (Zhang<em> </em>et al., 2014). The sequences were
 processed using QIIME2 v 2020.6 (Bolyen<em> </em>et al.,
 2019). We removed duplicated sequences and chimeras using the Vsearch
 (Rognes et al., 2016) plugin in QIIME2, and the molecular operational
 taxonomic units (MOTUs) at a 97% similarity threshold were clustered
 (Vamos et al., 2017). We removed sequences that occurred only in a single
 sample and sequences with frequencies &lt; 3. The MOTUs representing
 &lt; 0.1% of the normalized sequences for each sample were removed to
 prevent the generation of potentially erroneous results (Bokulich et al.,
 2013). Representative sequences of each MOTU were compared with the
 reference sequences in the BOLD database (https://www.boldsystems.org/)
 and the Genbank database (https://www.ncbi.nlm.nih.gov/genbank/) to obtain
 taxonomic information. The taxonomic identification followed the criteria
 of Aizpurua<em> </em>et al., (2018) and Alberdi et al.,
 (2018). Order-level taxonomic identifications were assigned at
 &gt; 95% similarity values; family-level identifications were assigned
 at &gt; 96.5%, and species-level identifications were assigned at 98%
 similarity. All of the identified species were checked manually. When one
 MOTU matched multiple species that shared the highest matching score, we
 downgraded the taxonomic resolution to the most common level. Those MOTUs
 not fulfilling the criteria or not matching any reference sequence were
 classified as unidentified. <strong>Statistical
 analysis</strong> For breeding time data, the
 first egg-laying date, incubation date, and hatching date were converted
 to Julian Days before analysis (1 April was defined as 1 each year (Fan et
 al., 2021)). For example, if the first egg-laying date was 1 May, this was
 recorded as 31. Generalized linear mixed models (GLMMs, Poisson
 distribution, log link, R package lme4) were used to examine differences
 in breeding time variables between tits and flycatchers, with species as a
 fixed variable. The first egg-laying date, incubation date, and hatching
 date were treated as response variables, and the year was treated as a
 random variable. We also calculated the breeding time overlap for each
 species. For example, the breeding time overlap for tits was calculated as
 the ratio of the co-breeding period with flycatchers to the total breeding
 period for tits. Due to the abnormal distribution of hatching rates
 (<em>P</em> &lt; 0.05), we used the Mann-Whitney U test to
 assess the differences between tits and flycatchers.
 For nest site data, Shapiro-Wilk tests were used to examine the
 normality of the distributions of nest site variables. If the
 characteristics were normally distributed, independent-sample t-tests were
 used to test for differences between tits and flycatchers. Otherwise,
 Mann-Whitney U tests were used (Appendix Table S1). For
 diet data, we computed and plotted the rarefaction and extrapolation
 curves of prey species richness based on Hill numbers (q = 0) using the R
 package iNEXT (Hsieh et al., 2016), developed for presence/absence data.
 The 95% confidence intervals (CIs) were obtained by a bootstrap method
 based on 1,000 replications. The relative read abundance (RRA) and percent
 frequency of occurrence (%FOO) were used to quantify the prey composition
 at the order and family levels, respectively (Deagle et al., 2019).
 Kruskal-Wallis tests were used to test for differences in the RRA between
 the two species. Chi-square tests were used to test for differences in the
 %FOO between tits and flycatchers; arthropods with frequency &gt; 2
 were selected for the calculation. Independent-sample t-tests were used to
 analyze the differences in the individual diet species richness between
 the two bird species that were represented as the average number of
 arthropod species (MOTUs) consumed by each individual of each species.
 Nonmetric multidimensional scaling (NMDS) with Bray-Curtis distance was
 used to examine the degree of diet similarity between the two bird
 species, and an analysis of similarities (ANOSIM) test with Bray-Curtis
 distance was used to examine the difference in diet composition between
 the bird species using the R package vegan (Ramette, 2007). Finally, the
 diet niche breadth of each species was calculated using a standardized
 Levins index (B<sub>A</sub>) (Hurlbert, 1978).
 To compare the niche occupied by the tit and flycatcher across
 multiple dimensions, we used a multivariate kernel density estimation
 method to measure interspecific niche overlap (Geange et al., 2011).
 Initially, we calculated niche overlap between the two species for each
 variable. Subsequently, we obtained the composite overlap across the three
 major ecological dimensions (breeding time, nest site, and diet) by
 averaging their corresponding variables. Finally, we obtained the overall
 niche overlap by averaging across all variables (Wang et al., 2021). We
 then used null model permutation tests to determine whether an observed
 overlap was significantly smaller than expected by chance. For each
 comparison, species labels were randomized 1000 times. The overlap between
 simulated kernel density distributions was compared with the observed
 overlap using t-tests. Significant differences indicated niche
 differentiation between the two species (Gotelli, 2000; Geange et al.
 2011). Except for the results of multivariate kernel
 density estimation (mean ± standard deviation), all other data were
 presented as mean ± standard error. All statistical analyses were
 conducted in R 4.1.2 (R Core Team 2022). The significance level was set at
 0.05.
description: # Multi-dimensional niche differentiation of two sympatric breeding
 secondary cave-nesting birds in Northeast China using DNA metabarcoding
 [https://doi.org/10.5061/dryad.dbrv15f8g](https://doi.org/10.5061/dryad.dbrv15f8g) We combined observations of breeding time, nest site, and diet to analyze the niche overlap and differentiation between two sympatric secondary cavity-nesting birds, the Japanese Tit *Parus minor* and the Yellow-rumped Flycatcher *Ficedula zanthopygia*. The results showed that 1) there were significant differences in the first egg laying date, length of the egg laying period, incubation date, and hatching date between tits and flycatchers, and the breeding time of flycatchers peaked later (about 30 days) than that of tits; 2) the two species had a large overlap in nest site factors, but the canopy coverage and shrub density of flycatchers were significantly higher than those of tits; 3) the two species had a large overlap in diet, and both largely relied on Lepidoptera (39.6% and 63.7% for tits and flycatchers, respectively), but flycatchers consumed significantly higher percentages of Lepidoptera, Diptera, and Coleoptera than tits. The results indicate that these two sympatric secondary cavity-nesting species have significant niche differentiation in breeding time, but little differentiation in nest site characteristics or diet. #### DATA & FILE OVERVIEW File List: nest_site_data.csv: Nest-site characteristics of boxes occupied by *Parus minor* and *Ficedula zanthopygia*. * Bird species: Parus minor or Ficedula zanthopygia - Year: Year of observation (2021 or 2022) * ID: Nest box ID - Tree species: Species of the nest tree * Nest height/cm: Height of nest above ground (cm) - Average diameter at breast height/cm: Mean DBH of 10 nearby trees (cm) * Nest tree height/m: Height of nest tree (m) - Nest box entrance: Orientation of entrance hole (e.g., N, E, S, W) * Canopy cover/%: Estimated canopy coverage above nest tree (%) - Number of tree species: Count of tree species within a 10 m radius * Number of trees: Count of trees within a 10 m radius - Entrance inclination: Angle of nest box entrance (°) * Shrub density/%: Estimated shrub cover (%) within 1 m radius - Average height of 10 shrubs/cm: Mean shrub height (cm) * Average diameter at breast height of 10 trees/cm: Mean DBH of 10 trees (cm) - Average height of 10 trees/m: Mean height of 10 trees (m) breeding_time_data.xls: Breeding timing data for *Parus minor* and *Ficedula zanthopygia*. * Bird species: Parus minor or Ficedula zanthopygia - First egg laying date: Calendar date of first egg * Incubation date: Date incubation began - Hatching date: Date of first hatchling * First egg laying date Julian day: Julian day converted with April 1 = 1 - Incubation date Julian day: Julian day for incubation start * Hatching date Julian day: Julian day for hatching MOTU_table.xlsx: *A*bundance table of Molecular Operational Taxonomic Units (MOTUs) generated from fecal metabarcoding. * MOTU_ID: Unique MOTU identifier * Subsequent columns: read counts for each sample (sample IDs correspond to fecal samples of individual birds) MOTUs_presence_absence_data.xlsx: Binary (1/0) presence–absence data for each MOTU across samples. * MOTU_ID: Unique MOTU identifier * Columns: sample IDs (1 = present, 0 = absent) hatching_rate.xlsx: Reproductive performance data. * Bird species: *Parus minor* or Ficedula zanthopygia * Clutch size: Number of eggs laid * Brood size: Number of nestlings hatched * Hatching rate: Ratio of brood size to clutch size prey_species_classified_at_the_order_level.csv: Relative occurrence of prey taxa classified at the order level. * species: *Parus minor* or Ficedula zanthopygia * id: Sample ID Araneae, Coleoptera, Diptera, Hemiptera, Hymenoptera, Isopoda, Lepidoptera, Neuroptera, Psocodea, Trombidiformes, Arachnida_others, Insecta_others, Malacostraca_others: Relative read abundance or occurrence frequency (%) for each prey order group.txt: Information on sample grouping for DNA metabarcoding analyses. * samples: Individual sample IDs corresponding to fecal or environmental samples * group: Species identity: *Parus minor* or *Ficedula zanthopygia* ## Sharing/Access information 1. Links to publications that cite or use the data: None. 2. Links to other publicly accessible locations of the data: None. 3. Was the data derived from another source? No.