DOI: 10.5061/dryad.w9ghx3fpv
title: Diversification patterns in Boeckella

description: Aim: We investigated evolutionary relationships and biogeographical
 patterns within the genus Boeckella to evaluate: 1) whether its current
 widespread distribution in the Southern Hemisphere is due to recent
 long-distance dispersal or long-term diversification; and 2) the age and
 origin of sub-Antarctic and Antarctic Boeckella species, with particular
 focus on the most widely distributed species: B. poppei. Location: South
 America, sub-Antarctic islands, maritime Antarctica, continental
 Antarctica and Australasia. Methods: To reconstruct phylogenetic patterns
 of Boeckella, we used molecular sequence data collected from 12 regions,
 and applied Bayesian and Maximum Likelihood analyses using multiple loci.
 We also estimated divergence times and reconstructed ancestral ranges
 using two different models of species evolution. Results: Phylogenetic
 analyses and divergence time estimates suggested that Boeckella originated
 on the Gondwanan supercontinent and initially split into two main clades
 during the late Cretaceous (ca. 80 Ma). The first clade diversified in
 Australasia and the second clade is currently distributed in South
 America, various sub-Antarctic islands and Antarctica. Dispersal from
 South America to the Kerguelen and Crozet archipelagos occurred during the
 Eocene/Oligocene (B. vallentini) and in the late Pliocene (B.
 brevicaudata), while South Georgia and the maritime Antarctic were likely
 colonized during the late Pleistocene (B. poppei). Main conclusions:
 Boeckella has a Gondwanan origin, with further diversifications after the
 physical separation of the continental landmasses. Extant populations of
 Boeckella from the Scotia Arc islands and Antarctic Peninsula originated
 from South America during the Pleistocene, suggesting that original
 Antarctic Gondwanan lineages did not survive repeated glacial cycles
 during the Quaternary ice ages. A continuous decline in the species
 accumulation rate is apparent within the genus since the early Eocene,
 suggesting that Boeckella diversification may have decreased due to
 progressive cooling throughout the Cenozoic era.
description: We extracted DNA from entire individuals using the DNeasy Blood
 & Tissue Kit (Qiagen, Hilden, Germany), with a modified protocol
 for small amounts of tissue (see Usage notes). Three different loci were
 amplified including a fragment of the mitochondrial cytochrome c oxidase
 subunit I (cox1) gene, the nuclear 28S rRNA gene and a segment spanning
 the Internal Transcribed Spacers 1 and 2 (ITS hereafter). For
 <b><i>cox</i>1</b> 2.5mL 10X Buffer (50 mM KCl, 10
 mM Tris-HCl, pH 8.0), 0.9 mL of 50 mM MgCl<sub>2</sub>, 200 mM
 dNTPs, 1mL of each primer (10 pg/mL), 0.5 mL BSA 10 mg/mL, 1 U Taq
 polymerase (Invitrogen), 14.5 mL of double-distilled water and 4 mL of
 DNA. Thermal cycling parameters for <i>cox</i>1 were modified
 from Scheihing et al., (2009), and included an initial denaturation step
 at 94ºC for 1 min, followed by 10 cycles at 94°C for 1 min, 40°C for 90
 sec and 72°C for 1 min, followed by 30 cycles at 94°C for 1 min, 46°C for
 90 sec and 72°C for 90 sec, and a final 10 min extension at 72ºC. For
 <b>28S</b> PCR reaction 2.5 mL 10X Buffer, 1 mL 50 mM
 MgCl<sub>2</sub>, 200 mM dNTPs, 1 mL of each primer (10
 pg/mL), 0.3 mL BSA 10 mg/mL, 1 U Taq polymerase (Invitrogen), 16.05 mL of
 double-distilled water and 2 mL of DNA. The PCR regime was as follows 94°C
 for 5 min, followed by 30 cycles at 94°C for 90 sec, 54°C for 90 sec and
 72°C for 2 min and a final 5 min extension at 72°C. For
 <b>ITS1/ITS2</b> the PCR reaction used 2.5 mL 10X Buffer, 0.3
 mL 50 mM MgCl<sub>2</sub>, 200 mM dNTPs, 1 mL of each primer
 (10 pg/mL), 0.5 mL BSA 10 mg/mL, 1 U Taq polymerase (Promega), 16.9 mL of
 double-distilled water and 2 mL of DNA. The PCR regime was 95°C for 5 min,
 followed by 43 cycles of 95°C for 90 sec, 55°C for 2 min and 72°C for 3
 min and a final 10 min extension at 72°C. PCR amplicons were purified and
 sequenced in both directions by Macrogen (Korea). Forward and reverse
 sequences were manually examined using Phred scores to ensure all
 sequenced bases matched and were of good quality. Contigs were assembled
 using GENEIOUS 10.2.2 (Kearse, Moir, Wilson, Stones-Havas, Cheung,
 Sturrock, Buxton, Cooper, Markowitz, Duran, Thierer, Ashton, Mentjies,
 & Drummond, 2012) and independently aligned using MUSCLE (Edgar,
 2004) with standard settings.   
description: <b><i>Kit QIAGEN DNA Easy Tissue </i></b><b>modify protocol</b> Use the whole individual and dry all the residual ethanol. Add <b>90 </b><b>m</b><b>L Buffer ATL.</b> Add <b>20 </b><b>m</b><b>L Proteinase K</b>. Mix thoroughly by vortexing, and incubate at 56°C overnight (6-10 hrs) on a rocking platform until the tissue is completely lysed. Vortex for 15 s. Add <b>12 </b><b>m</b><b>L Proteinase K</b> and incubate in a water bath at <b>60ºC</b> for <b>1h.</b> Vortex occasionally during incubation to disperse the sample every 15m. Add <b>100 </b><b>m</b><b>L Buffer AL</b> and <b>100 </b><b>m</b><b>L ethanol</b> (96-100%) and mix thoroughly by vortexing immediately after to yield a homogeneous solution. Pipette the mixture from step 6 (including any precipitate) into the DNeasy Mini spin column placed in a 2 mL collection tube (provided). Centrifuge at 8000 RPM for 1min. Discard flow-through. Add <b>250 </b><b>m</b><b>L Buffer AW1</b> and centrifuge at <b>8000RPM </b>for <b>1min. </b>Discard flow-through. Place the DNAeasy Mini spin column in a new 2 mL collection tube (provided), add <b>250 </b><b>m</b><b>L Buffer AW2</b>. Centrifuge for <b>3min at 14000RPM</b> to dry the DNAeasy membrane.<b> </b>Discard flow-through and collection tube. Place the DNAeasy Mini spin column in a clean 1.5 mL tube and add <b>50 </b><b>m</b><b>L Buffer AE</b> directly onto the DNAeasy Mini membrane. Incubate at room temperature for 1 min and then centrifuge for <b>1 min </b>at<b> 8000 RPM</b>. Repeat step 13 passing again the <b>50 </b><b>m</b><b>L flow-through</b> the membrane. This step leads to increased overall DNA yield.