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Xian Hou, Shengkai Pan, Zhenzhen Lin, Jiliang Xu, Xiangjiang Zhan. 2021: Performance comparison of different microbial DNA extraction methods on bird feces. Avian Research, 12(1): 19. DOI: 10.1186/s40657-021-00254-9
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As an important player during food digestion, gut microbiota has attracted much attention in diet adaptation studies in birds. Microbiota extracted from feces has been widely used as a proxy for gut microbiota. Although several methods have been developed for microbial DNA extraction, their performances in the bird feces have not beensystematacially evaluated yet.
In this study, we applied three DNA extraction methods (Qiagen, MoBio and Bead) to extract DNA from feces of three avian dietary guilds (granivore, omnivore and carnivore), sequenced V4 region of 16S rRNA gene for each extract and evaluated the performances of DNA yield, DNA integrity, microbial composition, cell lysis capacity and alpha diversity for the three methods on each dietary guild.
Bead method was thebest onthe performance of both DNA yield and DNA integrity regardless of dietary guild. In granivore, microbial relative abundance at both species and phylum levels, alpha diversityand cell lysis capacity were comparable among all methods. In omnivore, Qiagen had the best performance on alpha diversity, followed by Bead and MoBio. There were small variations on microbial relative abundance at both species and phylum levels among different extraction methods. MoBio exhibited the best performance on cell lysis capacity. In carnivore, considerable variations were found on microbial relative abundance at both species and phylum levels. Qiagen had the best performance on alpha diversity, followed by MoBio and Bead. MoBio had the highest cell lysis capacity.
DNA yield and integrity have no obvious impact on microbial composition, alpha diversity or cell lysis capacity. The microbiota results (e.g., microbial composition, cell lysis capacity, alpha diversity) obtained from different methods are comparable in granivorous avian species but not in omnivorous or carnivorous birds. Either method could be used in granivore microbiota studies. For omnivores and carnivores, we recommend Qiagen method when the research purpose is microbial diversity and MoBio when gram-positive bacteria is the research target.
Mate choice is one of the main sexual selection mechanisms and can lead to the evolution of conspicuous or even excessive phenotypic characteristics in animals (Andersson 1994). Bird songs tend to be long and complex vocalizations produced by male individuals during the breeding season (Catchpole and Slater 2008) and are prominent criteria in the mate choice (Searcy and Andersson 1986). Females generally prefer mates with a large sized repertoire, promoting the evolution of song complexity (Searcy 1992). In contrast, some songbirds have far simpler and less diverse vocalizations (Byers and Kroodsma 2009). The function and evolution of such "simple songs" and their relationship to sexual selection have attracted little attention (Cardoso and Hu 2011) and our understanding of mate choice of these species is therefore limited, besides its potential significance to the exploration of avian song evolution.
Generally in nature, conflicting pressures of sexual selection as well as in natural selection have long been viewed as a means to stabilize the evolution of sexually selected signals, such as ornaments and songs in birds (Andersson 1994; Gray and Cade 1999). Females can reap benefits both directly and indirectly through an appropriate mate choice (Møller and Thornhill 1998), e.g., getting better food resources, nest sites, nuptial gifts and also better genes for their offspring. Furthermore, parental investment strategies of both sexes may be influenced by their choice in mates, which in turn affects the survival of their offspring (Mitchell et al. 2007; Ligon and Hill 2010). For example, female Common Moorhens (Gallinula chloropus) compete for relatively fat males that are more persistent incubators (Petrie 1983), while female Common Stonechats (Saxicola torquata) prefer males with higher song rates, which apparently signals that they are more diligent in feeding offspring and in nest defense (Greig-Smith 1982). Male Great Tits (Parus major) with a large central black breast-stripe are also more likely to defend their brood (Norris 1990). Moreover, females can alter egg size (e.g. Cunningham and Russell 2000; Rutstein et al. 2004; Bonato et al. 2009) or feeding effort (e.g. Linville et al. 1998; Ligon and Hill 2010) according to the quality of their mates.
Most of the Phylloscopus warblers are accomplished singers with remarkable and impressive songs. In the Hume's Warbler (P. humei), however, only two simple songs have been reported (Baker 1997). They are described either as a double whistle note or as a drawn-out descending buzz. Furthermore, for P. h. mandellii, it had been discovered that this subspecies only issues the buzz song type (Fig. 1), but has five types of call (Bi 2004). It is still unclear why the songs of P. humei are so different from other apparently closely related Phylloscopus species (Baker 1997; Bi 2004).
Phylloscopus humei was originally considered as conspecific under the name of Yellow-browed Warbler P. inornatus (Voous 1977) but, based on acoustic, molecular, morphological and behavioral evidence, it is now accorded full species status (Irwin et al. 2001). Two disjunct subspecies are recognized: P. h. humei in India and P. h. mandellii in central China (Baker 1997). It is monomorphic, lacking obvious sexual ornaments but males, competing for territories early in the breeding season, display their pale wing-bars in P. h. humei (Marchetti 1993, 1998). Hitherto, only some basic descriptions about population status and breeding are available for the Chinese subspecies (Bi et al. 2009). In order to explore the possible relationships between mate choice and the simple song of P. humei, we conducted a field study of P. h. mandellii in the Lianhuashan Nature Reserve, Gansu, China. We specifically studied (1) preferences of females in their mate choice (considering both visual and song characteristics) and (2) whether the survival and development of their offspring is related to their mate choice.
We studied breeding P. h. mandellii birds at Shahetan station in the Lianhuashan National Nature Reserve (34°40′67″N, 103°30′84″E) during the April-July periods in 2009 and 2010. The elevation of the station is 2850 m asl. Mature mixed forests throughout the study area are dominated by spruce, fir, willow and birch trees (Sun et al. 2003). Local ground vegetation varies from sparse vegetation to dense shrubs of different species, as well as grasses, Arrow Bamboo (Sinarundinaria nitida), forbs and mosses (Sun et al. 2003).
We captured a total of 55 males using mist nets and marked them with unique combinations of colored leg bands to facilitate field identification. We captured male individuals as soon as possible when we detected their singing at the beginning of the breeding season (from late April to the middle of May). We opted for three types of measurements, i.e., body characteristics, ornamental and song characteristics to assess the relationship with female mate preferences. Specifically, we measured (1) bill length (exposed culmen), wing length, tail length and tarsus length as body characteristics using a centimeter ruler to the nearest 0.1 mm; (2) eyebrow size and wing-bar size (= length × width) as the ornamental characteristics using vernier calipers to the nearest 0.1 mm. We also recorded songs from differently marked males using a Sony WM-D6 recorder and a Sennheiser directional microphone. Song recording would be conducted at least 1 day after capture. A total of 139 recordings were analyzed using Avisoft SASLab Pro v. 4.52 applying the following settings: sampling frequency 22, 050 Hz, 16 bit, time resolution 5.8 ms and bandwidth 162 Hz. In order to remove background sounds (the noises and sounds of other birds), all recordings were filtered using the FIR high-pass filter in the software with a low frequency limit of 4.5 kHz before our analyses. We measured the following song parameters: duration (DUR), interval (INT), maximum amplitude (MA), distance from start to maximum amplitude (DSM), maximum frequency in the maximum amplitude (MAXMA), minimum frequency in the maximum amplitude (MINMA), frequency bandwidth in the maximum amplitude (FBMA), maximum frequency (MAXF), minimum frequency (MINF) and frequency bandwidth (FB).
We found nests by following females during the nest building period (mainly from late May to early June) and checked daily to measure the length, width and mass of newly laid eggs. In order to minimize disturbance when following females, we would check the nest after the female left. This bird has strong territorial defense behavior throughout the breeding period. In order to determine the ownership of the nest by the male, we would identify the territory of each male bird according to our field observation and as well check the color rings of male birds to verify the ownership during the incubation and/or nestling period. All nests laid eggs successfully. We calculated the egg volume as 0.457 × length × width2/1000 (Hoyt 1979). Following Marchetti (1998), we used the first egg date as a quantitative measure of female mate preference (i.e., females preferred the characteristics of males who supported the earliest possible nestings). Since the nestling period of P. h. mandellii is about 14 days (Bi et al. 2009), we measured the nestlings on Day 12 (bill length, wing length, tail length and tarsus length) to assess the development during the nestling period.
In order to identify the relationships among the characteristics of the body of males, egg size and fledging size, we applied a Generalized Linear Model (GLM) using R (version 2.14.1). Since multi-collinearity of independent variables can cause problems in regression models (Hosmer and Stanley 2000), we first calculated the bivariate correlations among different variables and then used a correlation coefficient threshold of 0.7 as suggested by Fielding and Haworth (1995). Fifteen variables were retained for further analysis, i.e., bill length, wing length, tail length, weight, tarsus length, eyebrow size, wing bar size, DUR, INT, DSM, MA, MAXMA, FBMA, MINF and FB. We conducted simple regression models for these variables and excluded nonsignificant variables (p > 0.05). Subsequently, we used a multiple regression model containing all significant variables and applied a stepwise backward procedure based on Akaike's information criterion (AIC, Akaike 1974) in order to reduce the number of variables.
During the 2 years of field work, we found 25 nests of the 55 captured males and another 18 nests of P. h. mandellii without capturing the male owners. From a total of 43 nests, we identified 40 with first egg laying dates, of which 18 reared at least two fledglings successfully. None of the nests were deserted in 2009, while two nests were deserted during the egg laying period and incubation period in 2010. This may be related to the harsh climate with continuous rain during 2010. Basic information about male characteristics, eggs and nestlings are presented in Additional file 1: Tables S1 and S2.
First egg dates were May 24 in 2009 and May 28 in 2010. In order to quantify the preference for a mate by a female individual, we defined these days as "1" and then transformed the first egg dates of the various nests into a numerical format (D). Five variables were significant (p < 0.05) in the simple regression analyses (Table 1). The DSM, FBMA and wing length have the highest explanatory power, with relatively large F values. The best multiple regression model only retained two of the five predictor variables entered (F2, 16 = 14.275, p < 0.001), i.e., DSM and wing length (Table 1), which represent the song and body size characteristics, respectively. The DSM affected the egg laying date positively, while the wing length was negatively correlated with D (Fig. 2). Furthermore, we found that the wing-bar size was significantly correlated with wing length (r = 0.513, n = 54, p < 0.001).
| Variables | Simple regression models | GLM | |||||||
| Estimate | SE | F1, 17 | p | Estimate | SE | t | p | ||
| Intercept | 19.803 | 16.913 | 1.171 | 0.259 | |||||
| Wing length | −9.544 | 2.804 | 6.808 | 0.002 | −4.650 | 2.625 | −1.771 | 0.096 | |
| Tail length | −5.821 | 2.782 | 1.002 | 0.048 | |||||
| DUR | 11.542 | 5.153 | 5.016 | 0.039 | |||||
| DSM | 13.951 | 2.936 | 22.57 | < 0.001 | 11.915 | 2.997 | 3.976 | 0.001 | |
| FBMA | 5.693 | 1.842 | 9.536 | 0.007 | |||||
| Only significant variables (p < 0.05) are listed. We report the estimated coefficients (Estimate), standard error (SE), t values and p values from GLM with step backward analysis | |||||||||
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Egg volume was correlated with egg mass (r = 0.929, n = 188, p < 0.001). Neither the mean egg mass (F1, 17 = 0.286, p = 0.600) nor egg volume (F1, 17 = 0.124, p = 0.729) were significantly correlated with first egg laying dates. However, the nestling bill length at Day 12 was negatively correlated with the first egg laying date (F1, 13 = 12.47, p = 0.004; Fig. 3a), suggesting that early nesting may confer benefits for the offspring. Further, nestling size was correlated with egg size, given that bill length was positively correlated with egg volume (F1, 16 = 10.11, p = 0.006; Fig. 3b).
The evolution of sexual phenotypic characteristics is affected not only by sexual selection, but also by natural selection (Andersson 1982). Highly developed secondary sexual characteristics generally require large investments in energy and attract predators. In either case, survival and both current and future reproduction may be adversely affected (Gray and Cade 1999).
As in P. h. humei (Marchetti 1998), we found that the criteria females use in their choice of a mate in P. h. mandellii include both visual signals (e.g. wing length) and song characteristics. We suggest that this phenomenon is also a joint action of natural and sexual selection (Höglund and Sheldon 1998; Kokko et al. 2002). In general, two hypotheses may be applied to explain the evolution of this type of mate choice and simple songs. First, Gilliard's 'transfer hypothesis' suggests that bright ornaments and other secondary sexual characteristics (including passerine songs) may be inversely related (Gilliard 1956); e.g., males of more dimorphic species have a dominant tendency to sing short songs in 56 North American wood warblers (Parulinae) (Shutler and Weatherhead 1990). However, P. h. mandellii obviously does not satisfy this hypothesis, for it has quite simple songs and its plumage is even less ornamental than that of other sympatric Phylloscopus spp., (e.g., P. kansuensis with a bright median crown stripe and yellow rump).
Generally, over-developed secondary sexual characteristics may require large amounts of energy and increase predation risks (Andersson 1994), which therefore should be more costly. Another proposed explanation is that females may judge male quality from quite subtle differences, such as small variations in male songs (Forstmeier et al. 2002) or slight variations in visual signals, such as eye ring pigmentation (Pérez-Rodríguez 2008), wing patch size (Török et al. 2003) or badge size (Griffith 2000). These types of criteria in females in mate choice can clearly reduce risk of predation. For P. h. mandellii females in particular, we suggest that these criteria of inconspicuous visual signals (e.g., longer wing and tail lengths) and short, inconspicuous songs may be partially related to their nesting behavior. As a ground nester, their inconspicuousness and monomorphism may be especially advantageous, as has been suggested for ground-nesting wood warblers as well (Shutler and Weatherhead 1990). Therefore, in order to reduce the probability of nest predation, P. h. mandellii may have evolved specific mate choice preferences in females for longer wings and smaller DSM (Fig. 2). Furthermore, given the statistically significant correlation between wing-bar size and wing length, we suggest that this bird may identify male size through the wing-bar, which should be rather easier to distinguish among individual males, as indicated in P. h. humei (Marchetti 1998).
With regard to song, we found that the first egg laying date was correlated with the parameters related to maximum amplitude, such as DSM, FBMA and MINMA. The most significant parameter was DSM, with early breeders having relatively short DSMs. Moreover, DUR was negatively correlated with first egg dates and DSM, suggesting that females prefer males whose songs are shorter with a faster increase in volume. Contrary to the study on P. h. humei by Marchetti (1998), we found that the song rate (=1/INT) was not correlated with the first egg date. This may be related to the multiple mate selection criteria and high plasticity in Hume's Warbler. Marchetti (1998) also found that the egg laying date was no longer correlated with song rate when wing-bar size was altered artificially. Furthermore, male songs have another important role in territorial defense. Catchpole (1983) has proposed that long songs may serve to attract mates while short songs are used for territorial defence. The shortness of territorial defence songs could be attributed to the need in males to hear the response of their competitors (Ince and Slater 1985). We accordingly suggest that the simple song of P. h. mandellii should play an important role in the defence of territory.
Finally, it should be stated that although females of P. h. mandellii in our study did not vary their investment in eggs in relation to the first egg laying date (i.e., mate choice), we obtained some evidence of faster growth in earlier hatched nestlings. This suggests that such chicks may have benefitted from higher rates of feeding or from better food. We observed that both parents bring food and feed the nestlings cooperatively. Therefore, we suggest that the female preference for larger males (i.e., longer-winged individuals) may be driven by the greater ability of such males to provision their young. Alternatively, these individuals may be more likely to occupy better territories with better food resources and/or better nest sites. Any or all of these features should play an important role in promoting the growth of their offspring and breeding success.
Additional file
Additional file 1: Table S1. Three types of measurements for captured males including body characters, ornamental and song characters. Table S2. The measurements of eggs and nestling.
NL and YHS designed the study. NL and JL collected the data and per‑ formed all analyses. NL and YHS drafted the manuscript. All authors read and approved the final manuscript.
This work was supported by the National Natural Sciences Foundation of China (Grant Nos. 31301886, 31272286). We sincerely thank Peipeng Luo and Yingxin Jiang for their great help in the field. We also thank Yi Tao, Chenxi Jia, Wenqin Zhong and Dan Strickland for their helpful comments.
The authors declare that they have no competing interests.
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Figures(4) / Tables(1)
| Variables | Simple regression models | GLM | |||||||
| Estimate | SE | F1, 17 | p | Estimate | SE | t | p | ||
| Intercept | 19.803 | 16.913 | 1.171 | 0.259 | |||||
| Wing length | −9.544 | 2.804 | 6.808 | 0.002 | −4.650 | 2.625 | −1.771 | 0.096 | |
| Tail length | −5.821 | 2.782 | 1.002 | 0.048 | |||||
| DUR | 11.542 | 5.153 | 5.016 | 0.039 | |||||
| DSM | 13.951 | 2.936 | 22.57 | < 0.001 | 11.915 | 2.997 | 3.976 | 0.001 | |
| FBMA | 5.693 | 1.842 | 9.536 | 0.007 | |||||
| Only significant variables (p < 0.05) are listed. We report the estimated coefficients (Estimate), standard error (SE), t values and p values from GLM with step backward analysis | |||||||||
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