KNUTSON & GOSLINER: NEW SPECIES OF GYMNODORIS NUDIBRANCHS 
FROM THE PHILIPPINES 
131 
ile water, 2.5 jiL lOX USB buffer, 1.0 |aL 25 mM MgCE, 1.0 gL each of 10 gM forward and reverse 
primer, 0.5 pL lOniM dNTPs, 1.5 pL, lOmg/mL BSA, 0.5 pL 1.25 units/pL HotStait Taq and 
between 2 pL to 5 pL of template DNA, depending on the concentration of the sample. Cycling 
parameters were generally as follows for COI: an initial denaturation step at 94°C for 3 min, fol¬ 
lowed by 94°C for 30 s, 47°C for 30 s, 72°C for 60 s for 35 cycles and a final extension step at 
72°C for 10 min. Following PCR, we visualized products on 1% agarose gel containing ethidium 
bromide. For reactions that successfully amplified the target gene fragment, we used an Exo-SAP- 
IT protocol ExoSAP-IT (usb.affymefrix.com) to purify products for cycle sequencing. 
Purified PCR products were cycle-sequenced using a ~10 pL reaction that contained 5.7 pL 
sterile water, 1.5 pL 5X Big Dye buffer, 0.3 pL 10 pM primer, 0.5 pL Big Dye 3.1, and 1-5 pL of 
purified PCR product, depending on the sample’s PCR band brightness during visualization. We 
cycle-sequenced all genes using a STeP Program (Platt et al. 2007) using an annealing temperature 
of50°C. 
Cycle-sequencing products were precipitated using a standard ethanol precipitation protocol 
and resuspended them in formamide. Samples were sequenced on the ABI Prism 3130 x 1 Genetic 
Analyzer at the Center for Comparative Genomics and the California Academy of Sciences. 
Sequence trace files were then assembled into contigs and edited them using the program 
Geneious version 6.0 (Drummond et al. 2011/We aligned the resulting consensus sequences in 
Geneious using the MAFFT Multiple Alignment vl. 1 (Biomatters Ltd) plugin with default settings. 
Geneious was then used to calculate the uncorrected p-distances between all the sequences. 
The COI sequences were uploaded to GenBank and the accession numbers appeal* in Appen¬ 
dix, Table 1. 
Results 
We were able to get COI sequences for 8 specimens of the two similar-looking taxa, 7 individ¬ 
ual sequences for one taxon and one of the second taxon. The uncorrected COI p-distances for the 
individuals within one taxonranged from 0.0% to 0.5%. Only one specimen amplified for the other 
taxon. The p-distances between this specimen and the other specimens ranged from 10.3% to 
11 . 1 %. 
Species Descriptions 
Gymnodorididae Odhner, 1941 
Genus Gymnodoris Stimpson, 1855 
Gymnodoris brunnea Knutson and Gosliner, sp. nov. 
Figures lA-B, 2-4 
Material examined.— Holotype: CASIZ 185943, one specimen, Anilao Pier, Anilao Har¬ 
bor, Balayan Bay, Batangas Province, Luzon Island, Philippines, 13°45'35.78"N 120°5533.56"E 
collected on April 30, 2011 by the members of the Hearst Philippine Biodiversity expedition, pre¬ 
served specimen 9,5 mm length. Paratypes; CASIZ 185946, one specimen, subsampled for DNA, 
preserved specimen 7.0 mm length with part of the “tail” missing, CASIZ 185968 one specimen 
dissected, subsampled for DNA, preserved specimen 9.2 mm length with part of the “tail” missing, 
CASIZ 185971, one specimen, preserved specimen 5.0 mm length, CASIZ 185972, one specimen, 
subsampled for DNA, preserved specimen 6.5 mm length with part of the “tail” missing, CASIZ 
185973, one specimen, dissected, CASIZ 185976, one specimen subsampled for DNA, preserved 
specimen ~8.0 mm length with part of the “tail” missing, all collected at the Anilao Pier, Anilao 
Harbor, Balayan Bay, Batangas Province, Luzon Island, Philippines, 13°4535.78"N 
120°5533.56"E on April 30, 2011 by the members of the Hearst Philippine Biodiversity expedi¬ 
tion. CASIZ 185979, one specimen, Matotonngil Point, Balayan Bay, Batangas Province, Luzon 
