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.

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 cox1 2.5mL 10X Buffer (50 mM KCl, 10 mM Tris-HCl, pH 8.0), 0.9 mL of 50 mM MgCl₂, 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 cox1 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 28S PCR reaction 2.5 mL 10X Buffer, 1 mL 50 mM MgCl₂, 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 ITS1/ITS2 the PCR reaction used 2.5 mL 10X Buffer, 0.3 mL 50 mM MgCl₂, 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. 

Kit QIAGEN DNA Easy Tissue modify protocol

1. Use the whole individual and dry all the residual ethanol.
2. Add 90 mL Buffer ATL.
3. Add 20 mL Proteinase K. Mix thoroughly by vortexing, and incubate at 56°C overnight (6-10 hrs) on a rocking platform until the tissue is completely lysed.
4. Vortex for 15 s.
5. Add 12 mL Proteinase K and incubate in a water bath at 60ºC for 1h. Vortex occasionally during incubation to disperse the sample every 15m.
6. Add 100 mL Buffer AL and 100 mL ethanol (96-100%) and mix thoroughly by vortexing immediately after to yield a homogeneous solution.
7. Pipette the mixture from step 6 (including any precipitate) into the DNeasy Mini spin column placed in a 2 mL collection tube (provided).
8. Centrifuge at 8000 RPM for 1min. Discard flow-through.
9. Add 250 mL Buffer AW1 and centrifuge at 8000RPM for 1min. Discard flow-through.
10. Place the DNAeasy Mini spin column in a new 2 mL collection tube (provided), add 250 mL Buffer AW2.
11. Centrifuge for 3min at 14000RPM to dry the DNAeasy membrane. Discard flow-through and collection tube.
12. Place the DNAeasy Mini spin column in a clean 1.5 mL tube and add 50 mL Buffer AE directly onto the DNAeasy Mini membrane.
13. Incubate at room temperature for 1 min and then centrifuge for 1 min at 8000 RPM.
14. Repeat step 13 passing again the 50 mL flow-through the membrane. This step leads to increased overall DNA yield.