146 
THE CORAL TRIANGLE: HEARST BIODIVERSITY EXPEDITION 
or unequal teeth. Myrianida is the only genus of Syllidae that reproduces by gemmiparous 
schizogamy (see Nygren, 2004 for hirther diagnostic characters). 
Material AND Methods 
The material examined was collected from less than 20 m depth in coral rubble at the “Dead 
Palm” dive site on the Calumpan Peninsula in Batangas Province at 13.69569°N, 120.88472°E. 
Rubble was collected by SCUBA during the Hearst Philippine Biodiversity Expedition 2011 by 
Bryan Rodriguez (University of Philippines Marine Science Institute) and Christina Piotrowski 
(California Academy of Sciences). 
Specimens were removed by soaking rubble in seawater for several hours and were sorted 
using a Meiji EMZ-13TR stereomicroscope, photographed, and preserved in 95% ethanol for 
DNA-sequencing. Specimens were studied under a Nikon Optiphot microscope with a differential 
interference contrast system (Nomarsky ) and an ocular micrometer, and were drawn by employing 
a camera lucida drawing tube. For scanning electron microscopy (SEM), the specimens were crit¬ 
ical point dried with an Emitech K850 Critical Point Dryer, gold-coated with a Q150T-S Turbo- 
Pumper Sputter Coater, and imaged using a Hitachi S-3000N electron microscope at SIDI (Servi- 
ciolnterdepartamental de Investigacion), Universidad Autonoma de Madrid (UAM). Width, 
excluding parapodia, was measured at the pro ventricle region. 
Genomic DNA was exti'acted using a DNeasy Tissue Kit (Qiagen) following protocols sup¬ 
plied by the manufacturer. Gene fragments of COl and IbSrDNA genes were amplified from the 
paratype specimen (MNCN 16.01/14734) of Myrianida puladilaw sp. nov. Universal primers ECO 
1490 (GGTCAACAAATCATAAAGATATTGG) and HCO 2198 (TAAACTTCAGGGTGAC- 
CAAAAAATCA) (Fohner et al. 1994) were employed for the COI fragment and the primers 
16SarL (CGCCTGTTTATCAAAAACAT) and 16SbrH (CCGGTCTGAACTCAGATCACGT) 
(Palumbi 1996) were used for 16S. PCR reactions were perfoimed using 3 ul of template DNA in 
25 ul reaction volumes using the following recipe for each gene fragment: COI: 2.5 ul lOX USB 
Buffer, 1.0 ul 25 niM MgCl 2 , 1.0 ul each of 10 uM primers, 0.5 ul of lOmM dNTP, 1.25 ul of 1.25 
U/ul HotStarTaq DNA Polymerase (Qiagen). 16S: 2.5 ul lOX USB Buffer, 1.5 ul 25 mM MgCU, 
0.2 ul each of 25 uM primers, 0.5 ul of lOniM dNTP, 1.25 ul of 1.25 U/ulTaq DNA Polymerase 
(Invitrogen). Both reactions were nm using a BioRad MyCyclerThermocycler. The temperature 
profile for COI was as follows: 94°C/180 sec; (94°C/30 sec; 48°C/30 sec; 72“C/60 sec) x 40 cycles; 
72°C/10 min; the temperature profile for 16S was: 94°C/180 sec; 50°C/120sec; 72°C/120sec; 
(94°C/35 sec; 50“C/30 sec; 72“C/40 sec) x 35 cycles). Two reactions were run for the template 
DNA using each recipe and temperature profile; products were then electrophoresed on an agarose 
gel and resulting bands were excised and combined into a single tube for each gene fragment and 
cleaned using a Zymoclean Gel DNA Recovery kit (Zymo Research). 
Twenty ul cycle sequencing reactions were perfomied for each primer using 3.0 ul of cleaned 
template DNA for each primer direction using the following protocols: (9.4 ul ultrapure H 2 O, 3.0 
ul 5X Big Dye Buffer, 0.6 ul 10 uM primer, 4.0 ul of Big Dye 3.1). The sequencing reaction tem¬ 
perature profile was as follows: 96°C/60 sec; (96°C/10 sec; 50°C/5 sec; 60°C/75 sec) x 15 cycles; 
(96°C/10 sec; 50°C/5 sec; 60^C/90 sec)x 5 cycles; (96°C/10 sec; 50°C/5 sec; 60°C/120 sec) x 5 
cycles. Sequencing products for each primer were precipitated using 125 mM EDTA and ethanol. 
Precipitated product was denatured, followed by analysis using an ABI 3130 Genetic Analyzer 
(Applied Biosystems), 
Reverse reads of sequence fragments were aligned by eye, trimmed, and edited using 
Sequencher 4.7 (Gene Codes Corporation). Overlapping sequence fragments were merged into 
