GONZALES & GOSLINER: NEW SPECIES OF PHILINE FROM THE INDO-PACIFIC 353 
ination of gizzard plates. Specimens of copulatory organs were mounted on stubs and air-dried or 
were photographed using automontage light microscopy. Hard structures were then coated with 
gold/palladium using a Denton Desk 11 vacuum sputter coater. Scanning electron micrographs were 
produced by a LEO 1450 VP scanning electron microscope. Specimens and dissected stmctures 
were deposited at the California Academy of Sciences in the Invertebrate Zoology Department col¬ 
lection (CASIZ). 
Molecular Methods 
Taxon sampling: Sampling of Philinidae specimens for the molecular study were all preserved 
in 70% EtOH, making it possible to perfonn molecular work on the tissue obtain sequences of the 
mithocondrial gene 16S. New molecular sequence data are provided for 21 specimens of 11 species 
of Philine and outgi*oup taxa (Table 1). Of the six species described here, five of the species were 
sequenced. Molecular data were not available fox Philine multipapillata, since the single specimen 
was preserved in formalin. The genus Scaphander was chosen as an outgroup based on its morpho¬ 
logical similarity and apparent close relationship to the genus Philine. Additionally, one specimen 
of Scaphander mundiis Watson, 1883, was sequenced to test the monophyly of Philine. The tissues 
came from specimens collected from various regions in the Indo-Pacific. The majority of speci¬ 
mens were found from deep waters off the Verde Island Passage, Philippines, but one additional 
species was collected from relatively shallow water from Panglao, Philippines, and another from 
shallow water in the Hawaiian Islands. Sequences of the 16S gene from 18 specimens of seven 
species in Philine were obtained from GenBank and were published by Krug etal. (2012) and used 
in analysis. They are therefore not included in Table 1. 
DNA extraction, amplification and sequencing: Genomic DNA was extracted from small 
pieces of foot tissue for most samples using QiagenDNeasy Tissue Kits. Amplification of DNA was 
conducted on BioRadsMyCycler™Thermocycler (software version 1.065, Bio-Rad Laboratories). 
Partial sequences of the mitochondrial genes 16S rRNA (485 bp) and 16Sar-L and 16Sbr-H 
(Palumbiet al. 1991) for one specimen for 16S PCR and sequencing we utilized internal primers 
developed by V. Knutson (personal communication). 
PCR amplifications were carried out in a 25 ml reaction volume including 2.5 ml of lOx PCR 
buffer, 0.5 ml dNTPs (10 mm stock), 1.0 ml MgCl (25 mm stock), 1.0 ml HotStartTaq (1.25 
units/ml)-Apex, 1.0 ml of each primer (25 mm stock), 15 pi H 2 O, and 2 ml of genomic DNA. The 
partial 16S amplifications followed the following parameters: an initial denaturing step at 94°C for 
3 min; 40 cycles of denaturing at 94°C for 30 s, annealing at 48°C for 30 s, amplifying at 72°C; 
and extension at 72°C for 5 min and 25°C for 5 min. 
PCR products were visualized on a 1.0% TBE agarose gel stained with ethidium bromide. 
PCR products were purified with ExoSAP-IT (USB Scientific) and then EtOH Precipitation. 
Cycle-sequencing reactions were performed using ABI Prism Big Dye Tenninator (Applied 
Biosystems) (total volume 10 ml) and analyzed using the automated sequencer ABI 3130 Genetic 
Analyzer (Applied Biosystems) in the Center for Comparative Genomics at the California Acade¬ 
my of Sciences (San Francisco, USA). All new DNA sequences have been deposited in Genbank 
(Table 1). 
Sequence alignment and analysis: The authenticity of the sequences was verified by BLAST 
comparisons. The sequences were edited and aligned using Genious 5.5 (Dmmmond et al. 2009) 
and checked by eye. 
Model selection and phylogenetic analyses: Phylogenetic data were analyzed using Maximum 
Likelihood with the program RAxML v7.04. RA xml was employed (Stamatakis, Hoover, and 
