1 A molecular approach to the study of birds’ DNA in faeces

2

3 David Pastor-Beviá*, Carlos Ibáñez, Juan Luís García-Mudarra, Javier Juste

4 Estación Biológica de Doñana, Consejo Superior de Investigaciones Científicas (CSIC),

5 Sevilla, Spain

6

7 *Corresponding author: David Pastor-Beviá

8 Address: Estación Biológica de Doñana, Consejo Superior de Investigaciones Científicas, c/

9 Américo Vespucio, s/n, 41092, Seville, Spain

10 Phone: +34669026480

11 E-mail: [email protected]

12

13 Running title: Birds DNA study in faeces.

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15 Keywords: giant noctule bat, faeces, DNA extraction, conservation, primer design, bird

16 DNA, amplification success.

17

18

19 Abstract

20

21 The molecular identification of prey in faeces is an efficient non-invasive technique to

22 study diet which requires both a satisfactory method of DNA extraction and the design of

23 specific primers to selectively amplify prey’s DNA. In this study we evaluated and compared

24 the efficiency of two total DNA extraction methods and five primer pairs for the molecular

1

25 identification of birds from scats, in particular from the giant noctule bat (Nyctalus

26 lasiopterus). A modified DNA stool Mini Kit of Qiagen was tested against a modified silica

27 method with a guanidinium thiocianate (GuSCN) applied after freezing and pulverizing the

28 samples. We also checked two published vertebrate- and bird-generalist primer pairs and three

29 bird-specific primer pairs designed by us (two pairs targeting the cytochrome b and one the

30 cytochrome oxidase subunit I genes) that amplified shorter DNA fragments. The results show

31 that pulverizing the scat remains before extraction was a very important step, presumably

32 facilitating access to the well preserved DNA located inside the rachis of the feathers. The

33 combination of our bird-specific designed primers showed a higher amplification rate than the

34 generalist primers and allowed successful bird identification from the feathers excreted by the

35 giant noctule bat in all the scat samples analyzed, independent of the conservation method

36 used (dried and frozen). These methodological improvements will allow not only the study of

37 the avian diet composition of the enigmatic giant noctule, but the extension of this

38 methodology to other bird predators such as raptors.

39

40 Introduction

41

42 Understanding food webs is a central question in ecology (Montoya & Solé, 2002).

43 Predator-prey interactions have long captured researchers’ attention because of the important

44 impacts that these interactions have both on prey and predator population dynamics and on

45 the functioning of the complete ecosystem (Lima, 1998). The selection of a particular prey is

46 a complex decision process that results from integrating factors such as prey size, density, and

47 availability (Griffiths, 1975; Symondson, 2002). Direct observations of feeding events are

48 often difficult in the field and diets are studied conventionally by the morphological

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49 identification of remains in faeces which many times is a challenging task due the effect of

50 mastication and digestion processes on these remains.

51 In recent times molecular and particularly, PCR-based techniques have proved to be

52 highly effective and versatile for studying diets and are likely to rapidly displace other

53 approaches (Symondson, 2002). PCR-based techniques target preys DNA in predators´

54 faeces. Nevertheless, detecting and amplifying degraded or semi-digested DNA is not always

55 an easy task mainly due to the presence in faeces of both reaction inhibitors and multiple

56 DNA templates. Besides, due to its higher proportion, the predator’s DNA can swamp the

57 result of the PCR amplification, although this problem can be normally avoided by using

58 species- or group-specific primers that exclusively target prey DNA sequences (King et al.,

59 2008). As a result, prey identification through the amplification of the prey´s DNA is now

60 possible even from highly degraded samples such as faeces, guts content or regurgitates

61 (Zeale et al., 2011). This possibility is changing the perspective of diet studies, and in fact the

62 approach has allowed identifying prey species in a variety of biological remains from spiders

63 (Agustí et al., 2003) to pinnipeds (Parsons et al., 2005), Macarony penguins (Deagle et al.,

64 2007) or bats (Clare et al., 2009; Zeale et al., 2011; Alberdi et al., 2012).Mitochondrial DNA

65 (mtDNA) has been widely employed in species identification due to its relative abundance

66 and high mutation rate, which facilitates the distinction between species, even between close

67 related ones (Brown et al., 1979). For this reason, mtDNA markers have been widely used in

68 diet studies, which usually rely on the partial or complete sequencing of genes such as

69 cytochrome b (cyt b) or cytochrome oxidase subunit I (COI). Particularly, the use of COI is

70 increasing owing primarily to its adoption by the Barcode for Life Consortium (Tobe et al.,

71 2010). DNA barcoding employs sequence diversity in short, standardized gene regions to aid

72 species identification and discovery in large assemblages of species. The Barcode of Life

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73 Data System (BOLD) — www. barcodinglife.org — provides an integrated bioinformatics

74 platform that supports all phases of the analytical pathway from specimen collection to a

75 tightly validated barcode library (Ratnasingham & Hebert, 2007).

76 Bats typically show a wide variety of diets that range from arthropods, vertebrates,

77 fruits, nectar and pollen, to leaves and even blood. This wide diversification of diet is striking

78 in comparison to other groups or even vertebrates (Altringham, 2011). In temperate

79 regions, bats are mainly insectivorous and they are major consumers of nocturnal

80 many of which are economically important pests (Agosta, 2002).

81 Diet studies of insectivorous bats traditionally relied on the morphological identification

82 in faeces of microscopic remains –primarily fragments of arthropod cuticles- resulting from

83 chewing prey into tiny pieces. These remains do not allow taxonomic identification normally

84 further than to Order, which usually provides only a gross estimate of the number and

85 diversity of insects eaten, hence providing only a reduced perspective on the real diet

86 (Whitaker et al., 2009; Zeale et al., 2011). On the other hand, the use of molecular techniques

87 has increased the accuracy of prey identification in bat diet studies even to the species level

88 (Zeale et al., 2011).

89 The giant noctule bat (Nyctalus lasiopterus) was found to be the first Palaearctic bat to

90 feed on flying passerines after the discovery of feathers in their faecal pellets (Dondini &

91 Vergari, 2000; Ibáñez et al., 2001). Small birds are preyed upon during their fall and spring

92 migrations (Ibáñez et al., 2001). This seasonal feeding switch from insects to birds was later

93 confirmed by stable isotopes analysis. The signature of the bat´s blood isotopic composition

94 peaked during Spring and Autumn, coinciding with the periods in which feathers appear in

95 the droppings and matching the birds migration patterns (Popa-Lisseanu et al., 2007).

96 Nevertheless, species composition of the avian diet of this bat remains still unknown since

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97 almost all the remains consist on little fragments of feathers (Dondini & Vergari, 2000) that

98 do not allow further morphological identification

99 Our objectives in this research are to 1) test if different conservation methods of faecal

100 samples and/or storage time affect DNA preservation, 2) develop an efficient protocol for

101 DNA extraction from bat faeces and 3) search for diagnostic mtDNA fragments that

102 amplifying only from birds’ DNA against other DNAs present in the bat scats, allow birds’

103 identification at species level.

104

105 Material and Methods

106

107 Collection, storage and preservation of faeces

108 For this study, we selected faecal pellets that showed presence of feathers from a total

109 of 47 giant noctule bats netted either over water courses in three points across Spain (La

110 Rioja, Malaga and Jaen), or captured as they returned to their tree roosts in a city-park in

111 Seville. Bats were individually kept in clean cloth bags and thereafter sexed, weighed,

112 measured, ringed and released at the site of capture. A total of 23 faecal pellets were

113 preserved by drying in paper bags at room temperature (11 collected in 2000 and 12 in 2005),

114 whereas another batch of 24 faecal pellets was preserved by freezing at -20ºC (13 collected in

115 2006 and 11 in 2009). All samples were processed in 2012.

116

117 DNA extraction

118 Total DNA extraction was performed in a sterile DNA laboratory. DNA was extracted

119 from about 0.01 – 0.05 g of faecal pellet unit using two different protocols: a) the Zeale et al.

120 (2011)’s method (based on a modified DNA stool Mini Kit of Qiagen) and starting by

5

121 washing the pellet, and b) the Rohland & Hofreiter (2007)’s silica method with a guanidinium

122 thiocianate (GuSCN), modified by first freezing the samples with liquid nitrogen and then

123 pulverizing them using 2 2.5 mm Ø steel balls. Both extraction methods were used in most of

124 scats (Table 2). DNA extracts were recovered with milli-Q water to a total volume of 60 µl.

125 Blanks without samples (one per extraction) were systematically included to detect possible

126 contaminations.

127

128 Primer design and testing

129 The resolution in bird identification of previously published general primers was tested

130 against newly designed ones both for fragments of cyt b and COI. We used the vertebrate

131 generalist primer pairs M13BC-FW/BCV-RV1 for a first PCR amplification, and M13/BCV-

132 RV2 for the products re-amplification (Alcaide et al., 2009) of a COI’s final fragment of 758

133 bp. In addition, the bird-specific primer pair Bird-F1/COIbirdR2, used previously for bar-

134 coding a bird community (Kerr et al., 2009), was used to amplify a COI 648 bp fragment. The

135 diagnostic resolution of these generalist and relatively long fragments were tested against

136 specific and short fragments of cyt b and COI obtained using newly designed primers. These

137 primers were designed from a selection of 81 homologous cyt b and 77 COI sequences from

138 86 birds downloaded from GenBank (http://www.ncbi.nlm.nih.gov/genbank) and BOLD

139 Systems (www.barcodinglife.org) and aligned with SEQUENCHER v. 4.9 (Gene Codes

140 Corporation Ann Arbor, MI USA). The selection was based on a list of potential bird preys of

141 the giant noctule in the Iberian Peninsula according to their weight (< 80 g) and wingspan (<

142 33 cm) and following Del Hoyo et al. (2004-2011). The final list of potential prey as

143 follows:7 Alaudidae, 3 Hirundinidae, 7 Motacillidae, 1 Troglodytidae, 9 Turdidae, 2

144 Muscicapidae, 2 Regulidae, 1 Cisticolidae, 23 Silviidae, 1 Paradoxornithidae, 5 Paridae, 1

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145 Remizidae, 1 Aegithalidae, 1 Sittidae, 1 Certhiidae, 1 Laniidae, 4 Passeridae, 10 Fringillidae

146 and 6 Emberizidae). Numbers of GenBank accession of all the sequences used are available in

147 Supplementary Material 1.

148 Primers were designed using the software Primer3 v. 2.3.2. (Koressaar & Remm, 2007) in

149 conserved regions and selected avoiding primer-dimer formation, self-complementarity, too

150 low melting temperature (Tm) and incorrect internal stability profile (Rychlik, 1995).

151 The sequences obtained with the new primers were first aligned with the programme

152 SEQUENCHER v. 4.9 and then inspected using PAUP 4.0.b10 (Swofford, 2001) to assure

153 that they represented unique haplotypes and that we were able to correctly distinguish each of

154 the species within the set of birds.

155

156 PCR amplification and sequencing

157 The 47 DNA extracts were amplified in a 28 µl total volume and a mixed composition

158 of: 3 µl 10 X NH4 buffer, 0.8 µl 50 mM MgCl2, 5 µg bovine serum albumin (BSA), 1.2 µg

159 dimethyl sulfoxide (DMSO), 0.2 mM dNTP mix, 0.75 µl 10 mM forward and reverse primer

160 and 0.16 µl 5 u/µl BIOTAQ DNA polymerase. Purified water was added to complete the 28

161 µl volume and 3 µl of DNA extract were added up to a final volume of 31 µl.

162 The PCR thermocycling included a 3 min initial denaturation step at 94 ºC followed by 35

163 cycles of 1 min at 94 ºC, 1 min at annealing temperatures between 45-55 ºC (depending on

164 primers), 1 min at 72 ºC followed by a final extension of 5 min. at 72 ºC. PCR products were

165 run and visualized on a 1% agarose ⁄ TBE gels stained with SYBR Safe, and purified by Big

166 Dye. Products were sequenced on an ABI 377 automated sequencer (PE Biosystems,

167 Warrington, UK) following the manufacturer´s protocols.

168

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169 Data analysis

170 In this study we investigated the effects on the amplification success of prey DNA in bat

171 scats of the time elapsed between faeces collection and DNA extraction (3 to 12 years),

172 conservation method (dried versus frozen) and DNA extraction method (two protocols).

173 Additionally, we checked the efficiency in the specific amplification of bird preys DNA of the

174 different pairs of primers targeting two mtDNA genes (cyt b and COI). To test the

175 significance of all these effects on DNA amplification, we performed Generalized Linear

176 Models (GLM) using the package “MASS” of the program R (Venables & Ripley, 2002).

177 Model construction followed a forward stepwise procedure and the selection was based on the

178 percentage of deviance explained and the Akaike’s Information Criterion (AIC), where

179 smaller AIC values suggesting a better fit to data. The analysis was performed in two steps: we

180 first tested for a determining influence on amplification by any of the selected factors using a

181 binomial GLM with all treatments and ‘DNA amplification’ as dependent variable. Then, we

182 tested for factors that influence the success in birds’ DNA amplification considering only

183 those treatments that yielded positive amplifications and using this time a negative binomial

184 GLM according to the distribution of the data. Amplifications were considered as ‘positive’

185 only when the resulting sequences were recognized as being from a bird. For this second

186 design, amplifications of bats’ DNA, of DNA from other organisms and ambiguous sequences

187 were considered ‘negative’ as the null amplifications since the possible prey –in case of being

188 amplified- couldn’t be identified unambiguously.

189

190 Results

191

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192 We selected 2 primer pairs for the cyt b and one primer pair for COI that amplified

193 fragments of 160 or 380 bp (Table 1). In the first analysis of DNA amplification success, most

194 of the variation (66.56%) was explained by a model that included all the factors (storing time,

195 conservation method, extraction protocol and pair of primers), although any of them had

196 significant influence by itself. For the second analysis, based on positive amplifications only,

197 the model with lower AIC and higher explanation of the variation (45.02%) included as

198 significant factors ‘conservation method’, ‘extraction protocol’ and their interaction, and

199 ‘primers selection’. The factor ‘storing time’ was the one explaining less percentage of

200 variation (less than 1%) and it was excluded of the model. The results showed statistically

201 significant differences between extraction methods depending of the conservation method

202 utilized. Thus, the modified silica extraction with pulverization was statistically significant

203 when the faeces had been dried, but not when they had been frozen (Z = -1.303, p = 0.192).

204 On the other hand, the amplification rate of our designed primers was always higher than the

205 amplification rate of vertebrate and bird generalist primers independent of the extraction

206 method whereas there were no statistically significant differences in amplification success

207 between the newly designed primers (Table 3, Figure 2).

208 All bird DNA fragments amplified were reliably identified to species level, indicating

209 that the sequences had a minimum of 4 bp differences between closely related species in the

210 case of the cyt b fragments (2.5% in CytbSPreyFW/RW and1.1% in CytbLPreyFW/RW, n=81

211 sequences), and 9 bp in the case of the COI fragments (1.2% in BC-FW-13/BCV-RV1, 1.4%

212 in Bird-F1/COIbirdR2 and 2.4% in COIPrey-FW/RW, n=77 sequences) according to the

213 absolute distance matrix obtained with PAUP for each primer set. These results indicated that

214 there is no risk of misidentifying species when we compare the sequences in GenBank.

215

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216 Discussion

217

218 Non-invasive techniques based on the amplification of genetic markers have proven to

219 be very effective in diet studies, offering significantly greater resolution in prey identification

220 than most of the traditional techniques (Symondson, 2002; King et al., 2008; Parsons et al.,

221 2005; Deagle et al., 2007; Dunshea, 2009; Zeale et al., 2011). Together with the DNA of the

222 consumed prey, faeces contain generally a mixture of DNAs originated from epithelial cells

223 that cover the intestinal walls of the predator, and from bacteria, fungi and parasites among

224 other sources. Nevertheless, the existence of multiple templates doesn’t affect to the sequence

225 determination since chimeras and contaminants were easily identified in the eye-inspection or

226 in the BLAST. In this study, bird DNA found in the excrements of noctule bats seems to be

227 better preserved within the feather’s rachis, which serves as a protective structure. This DNA

228 is less susceptible to the effects of gastric acids, degradation by external agents and the effect

229 of organisms (e.g. Haag et al., 2009; Farrell et al., 2000; Murphy et al., 2007; Brinkman et

230 al., 2010). Therefore, the amplification of relatively larger fragments (160-380 bp) than those

231 usually analyzed (100-250 bp) in diet studies (Pompanon et al., 2011) is more likely. In our

232 analyses, the GuSCN-based extraction method with previous pulverization has proven to be

233 more effective than the washing Qiagen method on dry-preserved scats. This result is

234 probably due to the fact that when pulverizing the faecal pellet, the within-rachis DNA is also

235 extracted and made available. Besides, it is expected that this DNA will be in better

236 conditions than other DNAs present in the faeces, and which amplification is highly

237 dependent on preservation and extraction methods (Frantzen et al., 1998; Piggott & Taylor,

238 2003; Prugh et al., 2005). In the case of the modified Mini Kit of Qiagen without

239 pulverization, only the DNA that remains more exposed is extracted and made available,

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240 whereas the DNA inside the rachis is not reached. This fact would explain why the

241 conservation method has a significant effect on the amplification success only in combination

242 with this extraction method. Thus, DNA amplification from material preserved frozen (and

243 not pulverized) yields better results -most probably due to less DNA degradation - than the

244 amplification from dried-preserved material. Finally, storing time does not significantly

245 influence DNA amplification success, probably because the period of time elapsed since the

246 collection of the samples and their study was not long enough for significant differences in

247 DNA’s breakdown and despite possible limitations in the sampling.

248 The mtDNA markers cyt b and COI have been used extensively by mammalogists and

249 ornithologists for species identification. At the beginning of this study, databases for cyt b

250 were more extensive for birds than those for COI although the later has been favored by

251 ornithologists due to the development of the DNA barcode and standardized reference

252 libraries. In our study both cyt b and COI showed similar resolution. Vertebrate and avian

253 generalist primers were only relatively efficient (25-38%) in the detection of avian DNA

254 within the bat’s faecal pellets. This was most probably due to their lower specificity and the

255 relatively large size (for degraded DNA) of the fragments that they amplified. It is well

256 known that smaller fragments survive better to digestion and are more detectable than larger

257 ones (eg. Sheppard & Harwood, 2005). Using the modified silica method extraction with

258 pulverization and the designed primers, the success percentage was between 81-89% and

259 none of the primers amplified bat’s DNA. These amplification success values are quite

260 satisfactory in relation to other studies. In fact, the percentage of predator DNA amplified

261 from fresh scat samples of captive jaguars was 87% (Haag et al., 2009), 76.8% from dried

262 scats of Andean cats (Napolitano et al., 2008), and 88% from scats of pine marten and stone

263 marten stored in ethanol or frozen (Ruiz-González et al., 2008).

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264 The three fragments obtained with our designed primers showed similar efficiency and

265 they can be used together for prey identification since the combination of two primer pairs

266 increases the amplification success rate up to 100%, although the pair CytbSPreyFW/RW

267 showed a wider range of amplification success percentages. We recommend combining the

268 COI primer pair (COIPrey-FW/RW) with CytbLPreyFW/RW to ensure that at least one

269 sequence can be found in the DNA databases. The bird species selection used to study

270 specificity is highly representative of the potential preys availability to the bats and since

271 there were no ambiguous haplotypes in the chosen fragments for any of the potential preys,

272 species identification will be unequivocal at least within the Iberian Peninsula.

273 Due to their specificity and high amplification rate, it will be highly useful to check

274 these new primers on a wider variety of test cases to establish their possible broader

275 applicability for studying the small bird component in the diet of other predators in the

276 Palaeactic, for example, of the other known bird-eating bats such as the great evening bat io

277 (Thabah et al., 2007), or the bird-like noctule Nyctalus aviator (Fukui et al., 2013). This

278 primer combination should work even for raptors such as the Eleonora´s falcon (Falco

279 eleonorae) and the sooty falcon (Falco concolor), which are the only two diurnal predators

280 that show a similar predatory strategy than the giant noctule bat in the Mediterranean region

281 (Ibáñez et al., 2001). So far, bird identification in the diet studies of these raptors is based on

282 the study of remains found near the nests, and this is not always possible (e.g. De León et al.,

283 2007). Our primers would help identify unambiguously the species to which all the remains

284 belong to.

285

286 In summary, the modified silica method with GuSCN binding solution (Rohland &

287 Hofreiter, 2007), in which samples are frozen by liquid nitrogen and then pulverized, turned

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288 out to be more effective than DNA stool Mini Kit (Qiagen) when the faeces are not fresh, due

289 to the fact that the DNA is extracted from feather remains protected of degradation inside the

290 rachis. When using the modified silica with GuSCN extraction protocol, the conservation

291 method used to store faecal samples as well as storing time do not affect amplification success

292 due to the pulverization of the sample. Our designed primers allow amplify satisfactorily

293 mtDNA fragments of birds consumed by the giant noctule bat and the identification of the

294 bird species for all the samples. These primers will allow us to study in detail the avian diet

295 composition of the poorly known giant noctule bat, unique in its partial carnivore diet in the

296 entire European continent, and could be used in the diet analysis of other bird predators.

297

298 Acknowledgements

299

300 Logistical support was provided by the ‘Laboratorio de Ecología Molecular’, at the

301 Estación Biológica de Doñana, CSIC (LEM-EBD). The EBD receives financial support from

302 the Spanish Ministry of Economy and Competitiveness, through the Severo Ochoa Program

303 for Centres of Excellence in R+D+I (SEV-2012-0262). This study was part of the MICINN

304 project CGL2009-12393 coordinated by J. Aihartza, UPV/EHU. We are grateful to Kent

305 Rylander for his help in the English usage. DPB acknowledges the financial support of the

306 Consejo Superior de Investigaciones Científicas (CSIC) through a JAE-PreDoc fellowship.

307

308

309

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415

416

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417 Figure Legends

418

419 Figure 1. Range, box-plots with 95% confidence intervals and mean values of the rate of

420 positive amplification success for the two extraction methods used.

421

422 Figure 2. Range, box-plots with 95% confidence intervals and mean values of the rate of

423 positive amplification success for the primers pairs used.

424

425

426

427 Supporting Information

428

429 Supplementary Material 1. List of species, numbers of GenBank accession of the sequences

430 used and mitochondrial region to which they belong.

431

432

433

434

435

436

437

438

439

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440 Table 1. Description and amplification conditions of the primer pairs designed. Forward

441 (FW) and Reverse (RW) primers are signalized following primer names.

442

443

Fragment Annealing Target gene Name Sequence size (bp) temperature (ºC) CytbSPrey (FW) AAT GGG ATT TTG TCG CAG TC Cyt b 160 52 CytbSPrey (RW) TCT CAG CCA TCC CCT ACA TC

CytbLPrey (FW) GAY AAA ATY CCM TTY CAC C Cyt b 380 47 CytbLPrey (RW) TGT TCD ACD GGY TGG CT

COIPrey (FW) CGA GCA GAR CTA GGC CAA CC COI 380 55 COIprey (RW) GCA GGC GGT TTT ATG TTG ATT GCT G 444

445

446

447

448

449 Table 2. Amplification success for each primer pair broken-down into extraction method

450 (with and without pulverization), conservation, and collection time. Positives: number of

451 faeces in which a bird sequence is amplified. Negatives: number of faeces in which there is no

452 bird DNA amplification (divided by samples without amplification and samples with

453 ambiguous identification).

454

455

456

457

458

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Without pulverization Whit pulverization CytbSPreyFW/RW CytbSPreyFW/RW

Positives Negatives Positives Negatives Method of conservation n Amplifications No amplify Amplify others n Amplifications No amplify Amplify others

Dry 2000 13 0 (0.0%) 13 0 11 9 (81.8%) 2 0

Dry 2005 12 4 (33.3%) 8 0 12 11 (91.7%) 1 0

Frozen 2006 8 0 (0.0%) 8 0 13 10 (76.9%) 3 0

Frozen 2009 9 1 (11.1%) 8 0 11 8 /72.7%) 3 0

TOTAL 42 5 (11.9%) 37 0 47 38 (80.6%) 9 0

CytBbLPreyFW/RW CytBbLPreyFW/RW

Positives Negatives Positives Negatives

n Amplifications No amplify Amplify others n Amplifications No amplify Amplify others

Dry 2000 13 0 (0.0%) 13 0 11 11 (100%) 0 0

Dry 2005 12 1 (8.3%) 11 0 12 9 (75%) 3 0

Frozen 2006 8 5 (62.5%) 3 0 13 12 (92.3%) 1 0

Frozen 2009 9 5 (55.6%) 4 0 11 10 (90.9%) 1 0

TOTAL 42 11 (26.2%) 31 0 47 42 (89.4%) 5 0

COIPreyFW/RW COIPreyFW/RW

Positives Negatives Positives Negatives

n Amplifications No amplify Amplify others n Amplifications No amplify Amplify others

Dry 2000 13 6 (46.2%) 7 0 11 11 (100%) 0 0

Dry 2005 12 10 (83.3%) 2 0 12 10 (83.3%) 2 0

Frozen 2006 8 5 (62.5%) 3 0 13 11 (84.6%) 2 0

Frozen 2009 9 7 (77.8%) 2 0 11 8 (72.7%) 3 0

TOTAL 42 28 (66.7%) 14 0 47 40 (85.1%) 7 0

Bird-F1/COIbirdR2 Bird-F1/COIbirdR2

Positives Negatives Positives Negatives

n Amplifications No amplify Amplify others n Amplifications No amplify Amplify others

Dry 2000 13 0 (0.0%) 13 0 11 3 (27.3%) 5 3

Dry 2005 12 0 (0.0%) 11 1 12 7 (58.3%) 4 1

Frozen 2006 8 5 (62.5%) 3 0 13 6 (46.2%) 6 1

Frozen 2009 9 3 (33.3%) 6 0 11 2 (18.2%) 5 4

TOTAL 42 8 (19.0%) 33 1 47 18 (38.3%) 20 9

BCFW-M13/BCRV1 BCFW-M13/BCRV1

Positives Negatives Positives Negatives

n Amplifications No amplify Amplify others n Amplifications No amplify Amplify others

Dry 2000 13 0 (0.0%) 7 6 11 5 (45.5%) 4 2

Dry 2005 12 0 (0.0%) 6 6 12 4 (33.3%) 2 6

Frozen 2006 8 2 (25.0%) 1 5 13 2 (15.4%) 7 4

Frozen 2009 9 0 (0.0%) 3 6 11 1 (9.1%) 6 4

TOTAL 42 2 (4.8%) 17 23 47 12 (25.5%) 19 16 459

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460 Table 3. Estimate, standard error (SE), Z-value and P-value of the factors that can influence

461 in the amplification success. Negative results were excluded. Significance codes: 0 ‘***’

462 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1.

463

Coefficients Estimate SE Z-value P-value

Intercept 4.4512 0.2006 22.192 < 2e-16 ***

CytbSPreyFW/RW -0.1117 0.2407 -0.464 0.64274

BirdF1/COIBirdR2 -0.7458 0.2462 -3.029 0.00245 **

COIPreyFW/RW 0.2488 0.2232 1.114 0.26514

BCFW-M13/BCRV1 -0.7439 0.2601 -2.86 0.00423 **

No pulverized:Frozen -0.3397 0.2073 -1.639 0.10126

Pulverized:Frozen -0.2522 0.1936 -1.303 0.19265

No pulverized:Dry -0.8443 0.2686 -3.143 0.00167 **

464

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