SUPPLEMENTARY MATERIAL
Peloso et al.
The Impact of Anchored Phylogenomics and Taxon Sampling on Phylogenetic
Inference in Narrowmouthed Frogs (Anura, Microhylidae)
Material and Methods
Laboratory Protocols
Sanger Sequencing (SGS).—Fragments targeted for PCR amplification and sequencing
were partial sequences of the mitochondrial ribosomal subunit 16S rRNA and
Cytochrome Oxidase I (COI), and partial sequences of the nuclear genes Brain-derived
Neurotrophic Factor (BDNF), Cellular Myelocytomatosis Oncogene - Exon 2 (CMYC),
Histone H3, Seven in Absentia homolog 1 (SIA1), and Tyrosinase. Targeted fragments
and primers used for PCR amplification and sequencing are those commonly used in
amphibian systematics. References for primers are given in Table 1 below.
PCRs were carried out in 25μl reactions (0.5-2.0μl of isolated DNA) using either
Fisher Taq/Buffer A (Fisher), or Illustra PuRe Taq Ready-To-Go PCR Beads (GE
Healthcare Life Sciences). For all amplifications, PCR programs included a denaturation
step of two minutes at 96°C, followed by 35 (mitochondrial) or 45 (nuclear) cycles of
amplification (95°C for 25s + 45-56°C for 25s + 72°C for 120s), with a final extension
step at 72°C for 5min. Standard annealing temperatures are given in Table S1. PCR
products were cleaned and desalted with an AMPure reaction (Agencourt Biosciences
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Corporation) on a Beckman Coulter Biomek 2000 robot. Cycle sequencing using BigDye
Terminators v. 3.0 (Applied Biosystems) was run in 10 μL reactions following the
protocol of Platt et al. (2007), and products were cleaned and desalted in a Cleanseq
reaction (Agencourt Biosciences Corporation) on the Biomek robot. Sequencing was
accomplished on an ABI 3730XL automated sequencer. Samples were sequenced in both
directions to check for sequencing errors and ambiguities. Contigs and sequence editing
were done in Geneious version 6.1.6 (Biomatters). Sequences were trimmed to the region
within the primers set. The 16S fragment was sequenced in two overlapping fragments
(primer pairs L2A/H10 + AR/BR) and sequences were trimmed to the region inside the
pair L2A/BR.
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Table 1. Primers used for PCR amplification (with annealing temperatures) and sequencing of loci employed in this study using
standard Sanger Sequencing techniques (see Material and Methods in main text).
Loci
Length
(bp)
Mitochondrial
Ribosomal
Subunit 16S
Cytochrome
Oxydase I
Brain-derived
Neurotrophic
Factor (BDNF)
658
Primer Sequence (5' > 3')
AR (part 2)
BR (part 2)
L2A (part 1)
H10 (part 1)
COI.PF-A
COI.PR-A
CGCCTGTTTATCAAAAACAT
CCGGTCTGAACTCAGATCACGT
CCAAACGAGCCTAGTGATAGCTGGTT
TGATTACGCTACCTTTGCACGGT
TTTCAACHAAYCAYAAAGAYATYGG
TANACTTCNGGGTGDCCAAARAATCA
BDNF.Amp.F1
ACCATCCTTTTCCTTACTATGG
700
BDNF.Amp.R1 CTA TCT TCC CCT TTT AAT GGT C
Cellular
Myelocytomatosis
Oncogene - Exon
2 (CMYC)
Histone H3
328
Seven in Absentia
homolog 1
397
Tyrosinase
Primer Name
cmyc1U
GAGGACATCTGGAARAARTT
cmyc-ex2 R
TCATTCAATGGGTAAGGGAAGACC
H3F
H3R
SIA1
SIA2
ATGGCTCGTACCAAGCAGACVGC
ATATCCTTRGGCATRATRGTGAC
TCGAGTGCCCCGTGTGYTTYGAYTA
GAAGTGGAAGCCGAAGCAGSWYTGCATCAT
TyrC
GGCAGAGGAWCRTGCCAAGATGT
TyrG
TGCTGGCRTCTCTCCARTCCCA
532
Standanrd
Annealing
Source
Temperature
48oC
Palumbi et al., 1991
Palumbi et al., 1991
o
48 C
Hedges, 1994
Hedges, 1994
45-48oC
Peloso et al. 2014
Peloso et al. 2014
o
52 C
van der Meijden et al,
2007
van der Meijden et al,
2007
o
52 C
Crawford (2003)
Wiens et al. (2005)
o
52 C
52oC
52-56oC
Colgan et al., 1999
Colgan et al., 1999
Bonacum et al., 2001
Bonacum et al., 2001
Bossuyt and
Milinkovitch, 2000
Bossuyt and
Milinkovitch, 2000
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