A Re-Evaluation of Phylogenetic Relationships Within Reed Warblers (Aves: Acrocephalidae) Based on Eight Molecular Loci and ISSR Profiles

Accepted Manuscript

A Re-evaluation of Phylogenetic Relationships within Reed Warblers (Aves: Acrocephalidae) Based on Eight Molecular Loci and ISSR Profiles

Tayebeh Arbabi, Javier Gonzalez, Michael Wink

PII: S1055-7903(14)00195-X DOI: http://dx.doi.org/10.1016/j.ympev.2014.05.026 Reference: YMPEV 4922

To appear in: Molecular Phylogenetics and Evolution

Received Date: 1 March 2014 Revised Date: 18 May 2014 Accepted Date: 21 May 2014

Please cite this article as: Arbabi, T., Gonzalez, J., Wink, M., A Re-evaluation of Phylogenetic Relationships within Reed Warblers (Aves: Acrocephalidae) Based on Eight Molecular Loci and ISSR Profiles, Molecular Phylogenetics and Evolution (2014), doi: http://dx.doi.org/10.1016/j.ympev.2014.05.026

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2 Warblers (Aves: Acrocephalidae) Based on Eight Molecular

3 Loci and ISSR Profiles

7 Tayebeh Arbabi, Javier Gonzalez, Michael Wink*

8 Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Heidelberg,

9 Germany

18 * Corresponding author. Address: Im Neuenheimer Feld 364, 69120 Heidelberg, Germany.

19 E-mail addresses: [email protected] (T. Arbabi), [email protected] (J.

20 Gonzalez), [email protected] (M. Wink).

22 ABSTRACT

23 Acrocephalidae is the most monomorphic family among passerines and has seen a long history

24 of different classifications and successive revisions. In this study, we evaluated the phylogenetic

25 relationships among 35 species of Acrocephalidae based on DNA sequences from five nuclear

26 loci (MB, ODC, LDH, FIB5 and RAG-1), three mitochondrial genes (CYB, ND2 and COI) and

27 genomic fingerprinting with ISSR-PCR. We could improve the resolution of phylogenetic

28 relationships among many species, but despite the use of 6280 nucleotides, some deep-level

29 relationships remain enigmatic. Lack of nodal support at some branches may be the result of

30 rapid radiation. The last common ancestor of this family dated for the Middle Miocene (14

31 MYA). In agreement with previous studies, we recovered the major clades of Acrocephalus,

32 Iduna (except I. aedon), Hippolais, Nesillas and Calamonastides. We accept the current

33 taxonomic position of Calamonastides gracilirostris as a monotypic genus and the inclusion of

34 Iduna natalensis and I. similis within Iduna but phylogenetic analyses based on mitochondrial

35 and nuclear genes as well as ISSR profiles did not support the position of I. aedon in Iduna.

36 Therefore, we resurrect the former genus Phragamaticola for this species in order to avoid

37 paraphyletic clades.

38 39 Keywords: Acrocephalidae, Acrocephalus, Hippolais, Iduna, aedon, ISSR. 40 41 42 43 44 45 46 47 48 49 50 51

52 1. Introduction

53 The avian family Acrocephalidae (Reed Warblers and allies) comprises 61 species (Gill

54 and Donsker, 2014) in the Palaearctic, Africa and Australasia. Most of the species share a similar

55 plumage pattern which represent identification challenges for ornithologists (del Hoyo et al.,

56 2006; Leisler and Schulze-Hagen, 2011). Despite numerous phylogenetic studies within this

57 family (Dickinson, 2003; Helbig and Seibold, 1999; Leisler et al., 1997), the phylogenetic

58 relationships remain partly speculative at the genus level. In a recent near complete phylogeny of

59 the Acrocephalidae, based on nucleotide sequences of the mitochondrial cytochrome b and three

60 nuclear genes (Fregin et al., 2009), five genera have been circumscribed: Acrocephalus, Iduna,

61 Hippolais, Nesillas and Calamonastides. These authors considered Chloropeta natalensis, C.

62 similis and Acrocephalus/Phragamaticola aedon being part of the genus Iduna (i.e. together with

63 species pallida, opaca, caligata and rama). Furthermore, Chloropeta gracilirostris is now

64 treated as belonging to the genus Calamonastides as previously proposed by Grant and

65 Mackworth-Praed (1940). Recent phylogenetic studies enabled to classify most taxa with

66 confidence but the phylogenetic relationships among a few groups as well as some species have

67 remained elusive (Fregin et al., 2009).

68 Here, we evaluated the phylogenetic relationships within the Acrocephalidae based on

69 DNA sequences from eight loci: five nuclear and three mitochondrial genes. Our alignment

70 consists of 6280 sites and DNA sequences from all loci were available for all species considered

71 in this study. Sequences from previously published papers were combined with our data set and

72 re-analyzed using standard phylogenetic reconstruction methods. Additionally, we made use of

73 Inter Simple Sequence Repeat (ISSR) PCR for genomic fingerprinting as a complementary

74 genetic marker since it has been successfully used in avian genetic studies (Fernandes et al.,

75 2013; Gonzalez and Wink, 2010; Gonzalez et al., 2008). We paid special attention to the

76 phylogenetic position of Iduna aedon, which had formerly been included in Acrocephalus.

78 2. Materials and methods

79 2.1. Sampling, amplification and sequencing

80 We included 35 species of the family Acrocephalidae in our multi-locus phylogenetic

81 analyses. These taxa comprise two individuals per taxon and two subspecies of A. scirpaceus

82 (Table S1). Based on previous phylogenetic studies (Fregin et al., 2009; Fregin et al., 2012), four

83 species, i.e. Pnoepyga pusilla, Locustella fluviatilis, Bradypterus baboecala and Megalurus

84 palustris were used as outgroups.

85 DNA was extracted from blood or tissue using a standard phenol chloroform protocol

86 (Sambrook et al., 1989). Eight genetic markers were sequenced and used for phylogenetic

87 reconstruction: three mitochondrial genes, i.e., cytochrome b (CYB; 1043 nt), NADH

88 dehydrogenase subunit 2 (ND2; 1077 nt) and cytochrome c oxidase subunit I (COI; 662 nt); and

89 five nuclear genes, i.e. myoglobin intron 2 (MB; 703 nt), ornithine decarboxylase, introns 6 and 7

90 (ODC; 776 nt), lactate dehydrogenase intron 3 (LDH; 513 nt), the fifth intron of beta fibrinogen

91 (FIB5; 562 nt) and a single exon of the recombination activating gene 1, (RAG-1; 944 nt). Primer

92 pairs used for amplification were: CYB: L14990/H16065 (Hackett, 1996; Kocher et al., 1989);

93 ND2: L5758/H5766 and L5216/H6313 (Sorenson et al., 1999); COI: PasserF1/PasserR1 and

94 ExtF/BirdR2 (Johnsen et al., 2010; Sheldon et al., 2009); MB: Myo2 (Slade et al., 1993)/Myo3F

95 (Heslewood et al., 1998); ODC: OD6/OD8r (Allen and Omland, 2003); LDH: b1/b4 and P5/P6

96 (Fregin et al., 2009); FIB5: Fib5/Fib6 (Marini and Hackett, 2002) and RAG-1: R17/R22 (Groth

97 and Barrowclough, 1999) and R50/R51 (Irestedt, 2001). In our analyses, we included sequences

98 available in GenBank and the new sequences generated in this study were deposited in GenBank

99 (see Table S1).

100 PCR amplifications were carried out in the following reaction mixture (total volume of

101 50 µL): 1.5 mmol/L MgCl2, 10 mmol/L Tris (pH 8.5), 50 mmol/L KCl, 100 µmol/L dNTPs, 0.2

102 units of Taq DNA polymerase (Bioron, Ludwigshafen, Germany), 200 ng DNA, and 5 pmol of

103 primers. PCR conditions consisted of an initial denaturation step at 94 °C for 5 min followed by

104 35 cycles at 94 °C for 1 min, annealing temperature for 1 min and 72 °C for 1 min; plus a final

105 extension step at 72 °C for 10 min. Optimal annealing temperatures were measured by a gradient

106 PCR in a Tgradient thermocycler (Biometra, Gottingen, Germany). Annealing temperatures

107 were: 50 °C (ND2), 51.5 °C (COI, LDH and FIB5), 52 °C (CYB), 53 °C (RAG-1) and 56 °C (MB

108 and ODC). PCR products were precipitated with 4 M NH4Ac and ethanol (1 : 1 : 6) and

109 centrifuged for 15 min (16,000 g). Sequencing was performed on an ABI 3730 automated

110 capillary sequencer (Applied Biosystems, CA, USA) with the ABI Prism Big Dye Terminator

111 Cycle Sequencing Ready Reaction Kit 3.1 (performed by STARSEQ GmbH, Mainz, Germany).

112 Sequencing primers correspond to the primers used in PCR amplifications.

113

114 2.2 ISSR genomic fingerprinting

115 ISSR–PCR was performed with 60 ng of template DNA in 25 µL reaction volumes

116 containing 10 pmol of the 5´-anchored microsatellite repeat primer [MW4 (GACA)4 or (GGTA)4],

117 0.1 mM each of dGTP, dCTP and dTTP, 0.045 mM dATP, 1 µCi (α-33P)-dATP (Perkin Elmer,

118 LAS, Rodgau, Germany), 0.6 units of Taq DNA polymerase (Bioron) and 2.5 µL of 10 ×

119 amplification buffer (10 mM Tris-HCl pH 8.5, 50 mM KCl and 1.5 mM MgCl2). DNA

120 amplifications were performed in a thermocycler Tgradient (Biometra, Goettingen, Germany).

121 Following the initial 5 min denaturation step at 94 °C, the program consisted of 38 cycles of 50 s

122 at 94 °C, 40 s at 50 °C, 2 min at 72 °C and 10 min at 72 °C for final elongation. Amplified

123 products were mixed with 8 µL bromophenol blue and run on a high-resolution denaturing

124 polyacrylamide gels 6 % (0.2 mm) for 4 h at 65 W (gel length 40 cm) containing 1 × TBE buffer.

125 The gels were dried and exposed for 2–5 days (depend on levels of radioactivity) to X-ray

126 hyperfilms (Kodak, Taufkirchen, Germany) and subsequently developed. Gel electrophoresis and

127 PCR reactions were performed twice for each sample to verify the repeatability of the results.

128

129 2.3. Alignment and phylogenetic analyses

130 Alignment was performed using Clustal W (Thompson et al., 1994) available in BioEdit

131 version 7.0.9.0 (Hall, 1999). All alignments were inspected and corrected visually. For nuclear

132 loci, heterozygous sites were coded as ambiguous. Indels were treated as missing data. The

133 concatenated alignment consisted of 6280 positions. Bayesian Inference (BI) phylogenetic

134 analyses were conducted in MrBAYES 3.1.2 (Ronquist and Huelsenbeck, 2003) and maximum

135 likelihood (ML) analyses in MEGA version 5.2 (Tamura et al., 2011). Eleven data sets were

136 analysed: all eight loci separately (Figs. S1-S8), combined mitochondrial genes (CYB, ND2 and

137 COI), five nuclear loci concatenated (MB, ODC, LDH, FIB5 and RAG-1), and all genes

138 combined in a single matrix. In multilocus analyses, the data were partitioned by locus. In

139 addition to the eight locus-specific partitions, the coding sequences were partitioned by codon.

140 Best evolutionary models that fit our data were selected according to a Bayesian Inference

141 Criteria (BIC) in JMODELTEST version 2.1.4 (Posada, 2008), which is presented in Table 1. BI

142 were carried out with two independent runs of 60 million generations each were conducted and

143 trees were sampled every 1000 generations, with the first 20 % of samples discarded as “burn-

144 in”. ML node support was assessed through 1000 bootstrap replicates. Posterior probabilities ≥

145 0.95 and bootstrap values ≥ 85% were considered as high support (Erixon et al., 2003).

146

147 2.4. Molecular clock based on mtDNA

148 Molecular clock analyses were conducted with three mitochondrial genes, i.e. CYB, ND2

149 and COI. Three loci were partitioned and unlinked for the analyses and each partition was

150 furthermore partitioned according to the three codon positions. We employed the lineage

151 substitution rate of 0.014 per lineage/million years (and corresponding standard deviations) for

152 CYB, 0.016 for COI and 0.029 for ND2 (Lerner et al., 2011). We made use of the model

153 TN93+I+G (Table 1) with a relaxed, uncorrelated lognormal clock and Yule process of

154 speciation as priors. MCMC analyses were run for 60 million generations with parameters

155 sampled every 1000 steps and 20% as “burn-in”. These analyses were performed in BEAST

156 version 1.8.0 (Drummond and Rambaut, 2007). Stationarity was assessed in TRACER 1.5 1

157 (Rambaut and Drummond, 2007).

158

159 2.5. ISSR analyses

160 ISSR-PCR bands were coded with 1 = band present and 0 = band absent. Clustering and

161 Neighbor joining tree reconstructions were conducted with Jaccard coefficient and 1000

162 bootstrap replicates in FAMD 1.31 (Schlüter and Harris, 2006). The tree was visualized using

163 TreeView version 1.6.6 (Page, 1996).

164

165 3. Results

166 3.1. Phylogenetic inferences

167 Our mitochondrial dataset for 72 individuals comprised 2782 characters. We translated

168 the nucleotide sequences to amino acid sequences using MEGA and did not find either stop

169 codons nor indels, suggesting that nuclear pseudogenes were not amplified (Allende et al., 2001).

170 We also obtained sequences of five nuclear genes for all species (Table S1). Including indels, the

171 concatenated mitochondrial and nuclear alignment comprised 6280 sites. Details are presented in

172 Table 1. From all reconstructed trees five reconstructions are shown here: combined

173 mitochondrial genes (Fig. 1), combined nuclear genes (Fig. 2), combined mitochondrial and

174 nuclear loci (Fig. 3) and a chronogram based on mitochondrial genes (Fig. 4). Finally, the

175 Neighbor-joining tree from ISSR fingerprints is illustrated in Fig. 6. All reconstructed trees

176 (Figs. 1–4) reveal six major clades in agreement with Leisler et al. (1997), Helbig and Seibold

177 (1999) and Fregin et al. (2009). Genus Acrocephalus with (1) subgenus Acrocephalus: large reed

178 warblers, (2) Calamocichla: Afrotropical/Malagasy reed warblers, (3) Calamodus: streaked reed

179 warblers plus unstreaked A. bistrigiceps and (4) Notiocichla: small unstreaked reed warblers. The

180 former genus Hippolais had been split into two clades: (5) Hippolais and (6) Iduna (Fregin et

181 al., 2009) which exclude I. aedon (formerly Acrocephalus aedon).

182 The clade of the subgenus Acrocephalus, which contains a large assembly of large reed

183 warblers (A. taiti, A. australis, A. stentoreus, A. orientalis and A. arundinaceus) from Palearctic-

184 Australasia, is strongly supported by both ML and BI analyses of mitochondrial, nuclear and

185 combined data set (PP ≥ 0.95 and ML bootstrap ≥ 85%). ML and BI analyses of combined and

186 mitochondrial trees supported the sister relationship of taiti/australis and stentoreus/orientalis;

187 but it is not the final position as many of the Pacific reed-warblers are missing in this analysis.

188 Subgenus Calamocichla consists of two well-defined groups in combined and

189 mitochondrial trees: one clade contains A. newtoni/A. sechellensis from Madagascar and the

190 Seychelles and the other includes A. gracilirostris as sister to the A. rufescens/A. brevipennis

191 clade. The exception is in nuclear tree where A. gracilirostris clustered with A. newtoni/A.

192 sechellensis.

193 The subgenera Acrocephalus and Calamocichla cluster as sister groups in all

194 reconstructed trees with high support (PP ≥ 0.95 and ML bootstrap ≥ 85%). The other larger reed

195 warbler, A. griseldis, occupies a basal position in relation to other large reed warblers. Due to

196 this particular phylogenetic position, it had been nominated as subgenus ‘‘Indeterminate” by

197 Helbig and Seibold (1999). In the ML of the all combined trees, as well as BI of nuclear genes,

198 the sister relationship of A. griseldis and subgenera Calamocichla/Acrocephalus receives high

199 support.

200 Four striped reed warbler species of the monophyletic subgenus Calamodus (A.

201 melanopogon, A. paludicola, A. bistrigiceps, and A. schoenobaenus) formed a well supported

202 clade while the phylogenetic position of this clade in the tree and the relationships within this

203 group remains uncertain: A. bistrigiceps as sister taxon to the melanopogon/paludicola pair; and

204 schoenobaenus is basal of all of them. These relationships were well supported in combined and

205 mitochondrial trees. In ML of nuclear tree the same relationship was found but with low support.

206 In BI of the nuclear gene tree, melanopogon/bistrigiceps and paludicola/schoenobaenus cluster

207 as sister groups with moderate support (not shown). Fregin et al. (2009) found different

208 relationships: In the combined nuclear tree and complete concatenated dataset bistrigiceps

209 clustered as a sister of paludicola, and melanopogon the sister of schoenobaenus but without

210 high bootstrap support.

211 The subgenus Notiocichla consists of two groups: in one clade A. baeticatus is the sister

212 species of the A. scirpaceus complex, followed by A. palustris and A. dumetorum which occupies

213 a basal position within this group. The second group comprises the A. concinens/A. tangorum

214 clade and the sister A. agricola. These relationships were well resolved in our analyses while

215 previous studies did not provide strong evidence for these associations. Fregin et al. (2009)

216 included A. orinus and found a sister relationship with dumetorum.

217 In ML analyses based on combined and nuclear datasets, Calamodus clusters with

218 Notiocichla, as proposed by Fregin et al. (2009), although the support for this relationship is low.

219 Leisler et al. (1997) and Helbig and Seibold (1999) did not obtain conclusive evidence regarding

220 the association between Notiocichla and Calamodus.

221 Uniting all the previous groups in the genus Acrocephalus as proposed by Fregin et al.

222 (2009) received various support in our analyses (Fig. 1–3) and also detected by ISSR analysis

223 (the subgenus Calamocichla was not included). Among the single trees, LDH and FIB could not

224 group them in one clade (Figs. S7 and S8).

225 The Hippolais clade (according to Fregin et al., 2009) was well supported in all analyses;

226 it consists of two well-supported sister clades: 1) H. languida/H. olivetorum, and 2) H.

227 polyglotta/H. icterina.

228 The newly formed genus Iduna splits into the following sister groups with high bootstrap

229 support in all analyses: 1) caligata/rama and 2) natalensis/similis (in exception of nuclear tree)

230 with pallida and opaca. The inclusion of I. natalensis and I. similis within Iduna as proposed by

231 Fregin et al. (2009) is robustly supported in our analyses. They were previously placed in the

232 genus Chloropeta (Sibley and Monroe, 1990). In the mitochondrial, nuclear and combined

233 phylogenetic tree the posterior probability is 1.0, bootstrap values vary between 98–100% and in

234 the Neighbor-joining tree based on ISSR data, the given support is 90%; which is a high support.

235 The exception is Iduna aedon (Fregin et al., 2009): its placement within the Iduna clade was

236 only supported by analyses based on nuclear genes, with a posterior probability of 0.65 and

237 bootstrap support of 83%. Among the individual gene trees, only ODC (PP = 1; ML = 91%) and

238 LDH (ML = 64%) showed the relationship and none of the mitochondrial genes supported this

239 association. The ISSR genomic fingerprint clustering analysis also revealed that aedon is not a

240 member of Iduna (Fig. 6) because it does not share most of the informative ISSR bands with

241 other Iduna taxa nor Acrocephalus.

242 The position of Calamonastides gracilirostris (Fregin et al., 2009; Grant and Mackworth-

243 Praed, 1940) remains uncertain. The analyses based on mitochondrial and combined datasets

244 revealed a group containing C. gracilirostris and I. aedon with very low support. The ISSR

245 analysis also found it in an isolated position.

246 Nesillas typica from Madagascar was basal to all the other clades and this association was

247 supported in BI with high posterior probabilities as well as ISSR Neighbor-joining tree. Among

248 the single trees ND2 (PP = 96; ML = 85%) and ODC (PP = 88) showed the same results while in

249 the other trees, it has ambiguous positions.

250 The chronogram based on a coalescent multi-locus analysis of three mitochondrial genes

251 revealed that most of the speciation events might have occurred in the Middle Miocene [14

252 million years ago (95% Highest Posterior Density: 11.87–16.34)] to Early Pleistocene [0.78

253 million years ago (95% HPD: 0.49–1.11); Fig. 4].

254

255 3.2. ISSR

256 ISSR-PCR fingerprints obtained with the primers (GACA)4 and (GGTA)4 from 22 taxa of

257 Acrocephalidae (two individuals per taxon with the exception of Iduna opaca ) are shown in Fig.

258 5. Profiles were more diverse with the (GGTA)4 primer than (GACA)4; however, a series of genus

259 or subgenus-specific ISSR products were obtained. In total, 45 clearly identifiable bands were

260 available for analysis. The ISSR tree is congruent with our phylogenetic analyses based on

261 nucleotide sequences. The major clades within the genus Acrocephalus (the subgenus

262 Calamocichla was not included) and the distinction between Hippolais and Iduna are also

263 detected by ISSR analysis; and in agreement with the sequence data, Iduna aedon did not cluster

264 within Iduna (Fig. 6).

265

266 4. Discussion

267 We used nucleotide sequences from eight loci, a number of markers close to the optimal

268 amount of genes suggested by Maddison and Knowles (2006), and ISSR fingerprints, in order to

269 reconstruct a phylogeny for 35 species of Acrocephalidae. Despite the use of 6280 nucleotides,

270 some relationships and several deep nodes in our phylogenetic trees were not well-supported.

271 The ISSR fingerprinting evidence did not resolve the basal polytomy in the phylogeny of

272 Acrocephalidae; however, it supports several clades retrieved by DNA sequence analyses.

273 The association between Chloropeta natalensis/C. similis (del Hoyo et al., 2006;

274 Dickinson, 2003; Sibley and Monroe, 1990; Wolters, 1982) and warblers of the genus Iduna

275 (Fregin et al., 2009), was supported by high bootstrap values in Ml and BI analyses as well as

276 ISSR fingerprints.

277 We accept the current taxonomic position of Calamonastides gracilirostris as a

278 monotypic genus, due to the unresolved position in the tree. However, this species clustered with

279 Calamocichla in our BI tree based on nuclear genes where it received high bootstrap support.

280 This relationship has been previously proposed by Grant and Mackworth-Praed (1940), based on

281 morphological similarities; but due to low bootstrap support in all the other trees, the position of

282 this taxon remains uncertain.

283 Iduna aedon (Fregin et al., 2009), also regarded as Phragamaticola aedon (Wolters,

284 1982) or Acrocephalus aedon (Sibley and Monroe, 1993), can be found in differing positions in

285 reconstructed phylogenetic trees with poor bootstrap supports. The ISSR profiles (Fig. 5) of

286 aedon also show substantial differences as compared with all other species. Recently, several

287 authorities (e.g. Gill and Donsker, 2014) transferred aedon from the monotypic genus

288 Phragamaticola to the genus Iduna following Fregin et al. (2009), although a recently published

289 reed warbler monographs (Kennerley and Pearson, 2010; Leisler and Schulze-Hagen, 2011) left

290 it in Phragamaticola. Iduna aedon has intermediate similarities. Based on large body size, it

291 might be supposed to belong to the Acrocephalus; but its egg coloration is similar to Hippolais

292 and Iduna natalensis (Schönwetter, 1979). However, it has many unique characters such as long

293 tail with narrow feathers, thick bill and lack of supercilium (Helbig and Seibold, 1999),

294 indicating a long period of genetic isolation which agree with the molecular analyses presented

295 here. Leisler & Schulze-Hagen (2011) have made a strong case to keep it in Phragamaticola by

296 showing that aedon differs not only in morphology, but also in a number of other traits, such as

297 nest construction and song from all other family members. Their song and nest cluster analyses

298 clearly show that it is not close to the Iduna group. Based on all these pieces of evidence, we

299 recommend the exclusion of aedon from Iduna and we propose to resurrect the previous status

300 for this species: the monotypic genus Phragamaticola.

301 Estimated divergence times suggest that the Old World warblers shared a common

302 ancestor 14 million years ago in the Middle Miocene. This relative young evolutionary time

303 (Beresford et al., 2005) of this groups and a subsequent rapid radiation make the systematics of

304 this diverse family a challenge (Helbig and Seibold, 1999).

305

306 Acknowledgments

307 The authors are grateful to Gerhard Nikolaus, Hans-Hinrich Witt, Rolf Klein, Dietrich

308 Ristow, Bernd Leisler and Martin Flade for providing samples. We also thank IPMB assistants

309 Heidi Staudter and Hedwig Sauer-Gürth for their help in the laboratory.

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413 Table legend

414 Table 1. Number of nucleotides, polymorphic sites (without outgroups) and the best

415 evolutionary model for each partition and combined data set.

416

417 Figure legends

418 Fig. 1. Bayesian inference tree based on concatenated mitochondrial CYB, ND2 and COI. BI

419 posterior probabilities and ML bootstrap values indicated above and below the branches,

420 respectively. Asterisks indicate posterior probability = 1.00 or bootstrap = 100%. Branches with

421 less than 50% support have been collapsed. Terminal branches contain two individuals per

422 species. Species names and genera at the right column follow Fregin et al. (2009) and subgenera

423 within Acrocephalus are indicated on the left side.

424

425 Fig. 2. Bayesian inference tree based on nuclear genes concatenated (MB, ODC, LDH, FIB5 and

426 RAG-1). Support values as in Fig. 1. Branches with less than 50% support have been collapsed.

427 Terminal branches contain two individuals per species. Species names and genera on the right

428 column follow Fregin et al. (2009) and subgenera within Acrocephalus are shown on the left

429 column.

430

431 Fig. 3. Phylogenetic tree based on a combined dataset (CYB, ND2, COI, MB, ODC, LDH, FIB5

432 and RAG-1) analysed by Bayesian inference. Support values marked as in Fig. 1. Branches with

433 less than 50% support have been collapsed. Terminal branches include two individuals per

434 species. Species names and genera in the middle follow Fregin et al. (2009), left column

435 indicates subgenera within Acrocephalus and genera suggested by the authors in bold.

436

437 Fig. 4. Chronogram of Acrocephalidae reconstructed based on Bayesian inference of

438 concatenated mitochondrial CYB, ND2 and COI. Analysis implemented in BEAST, using

439 relaxed, uncorrelated lognormal clock (mutation rates of 0.014 substitutions/site/lineage/million

440 years for CYB, 0.016 for COI and 0.029 for ND2), TN93+I+G as evolutionary model, Yule

441 process of speciation as priors and codon position partitioned nucleotide substitution model. Blue

442 bars represent 95% highest posterior density intervals for the node ages. Posterior probabilities

443 are indicated at the nodes and branches with less than 50% support have been collapsed. Branch

444 lengths are in millions of years ago. Species names follow Fregin et al. (2009).

445

446 Fig. 5. ISSR profiles using the primers (GACA)4 (top) and (GGTA)4 (bottom). Numbers

447 correspond to the following groups and species: 1–3 subgenus Acrocephalus (A. arundinaceus,

448 A. orientalis, A. australis); 4–6 Calamodus (A. schoenobaenus, A. melanopogon, A. paludicola);

449 7–9 Notiocichla (A. agricola, A. scirpaceus, A. palustris); 10–13 Hippolais (H. languida, H.

450 olivetorum, H. polyglotta, H. icterina); 14 I. aedon; 15–20 Iduna (I. rama, I. caligata, I. pallida,

451 I. opaca, I. natalensis, I. similis); 21 Calamonastides gracilirostris; and 22 Nesillas typica.

452

453 Fig. 6. Neighbor-joining tree of 22 selected taxa of Acrocephalidae, reconstructed using 45

454 polymorphic ISSR bands, Jaccard coefficient of similarity and 1000 bootstrap replicates. Species

455 names and genera on the right column follow Fregin et al. (2009) and subgenera within

456 Acrocephalus are indicated on the left column.

457

458 Supplementary Material

459 Table S1. Studied taxa in alphabetical order, locality, IPMB (Institute of Pharmacy and

460 Molecular Biotechnology) numbers and GenBank accession numbers for every gene. Sequences

461 that are new to this study are in bold. Asterisks indicate the sequences have previously done in

462 IPMB lab (Leisler et al., 1997).

463

464 Fig. S1. Phylogenetic tree based on mitochondrial CYB. BI posterior probabilities and ML

465 bootstrap values indicated above and below the branches, respectively. Asterisks indicate

466 posterior probability = 1.00 or bootstrap = 100%. Branches with less than 50% support have

467 been collapsed. Species names follow Fregin et al. (2009).

468 18

469 Fig. S2. Phylogenetic tree based on mitochondrial COI. Support values as in Fig. S1. Branches

470 with less than 50% support have been collapsed. Species names follow Fregin et al. (2009).

471

472 Fig. S3. Phylogenetic tree based on mitochondrial ND2. Support values as in Fig. S1. Branches

473 with less than 50% support have been collapsed. Species names follow Fregin et al. (2009).

474

475 Fig. S4. Phylogenetic tree based on nuclear MB. Support values as in Fig. S1. Branches with less

476 than 50% support have been collapsed. Species names follow Fregin et al. (2009).

477

478 Fig. S5. Phylogenetic tree based on nuclear ODC. Support values as in Fig. S1. Branches with

479 less than 50% support have been collapsed. Species names follow Fregin et al. (2009).

480

481 Fig. S6. Phylogenetic tree based on nuclear RAG-1. Support values as in Fig. S1. Branches with

482 less than 50% support have been collapsed. Species names follow Fregin et al. (2009).

483

484 Fig. S7. Phylogenetic tree based on nuclear LDH. Support values as in Fig. S1. Branches with

485 less than 50% support have been collapsed. Species names follow Fregin et al. (2009).

486

487 Fig. S8. Phylogenetic tree based on nuclear FIB5. Support values as in Fig. S1. Branches with

488 less than 50% support have been collapsed. Species names follow Fregin et al. (2009).

489

490

491

492

493

494

495

496 23

498 25

500

501

502

Gene Nucleotides Polymorphic sites Model of evolution (percentage) CYB 1043 421 (40.36 %) TN93+I+G ND2 1077 506 (46.98 %) TN93+I+G COI 662 237 (35.80 %) TN93+I+G MB 697 78 (11.19 %) HKY+G ODC 774 107 (13.82 %) HKY+I LDH 492 93 (18.90 %) HKY+G FIB5 562 97 (17.26 %) HKY RAG-1 944 44 (4.66 %) TN93+G Mitochondrial loci 2782 1164 (41.84) TN93+I+G Nuclear loci 3498 419 (11.98) HKY+G Combined dataset 6280 1583 (25.21) TN93+I+G 503 504

505

506

507 Highlights 508 509 • We investigate phylogenetic relationships within Acrocephalidae. 510 • We analyze 3 mitochondrial and 5 nuclear loci as well as ISSR. 511 • We reject the inclusion of I. aedon in genus Iduna. 512 • We propose the genus Phragamaticola for aedon.

513 514