Laidig et al.: Descriptions and growth of larval and juvenile Sebastes wilsoni 



453 



developmental stages. Further, we examine otolith ra- 

 dius at time of larval extrusion to separate pygmy rock- 

 fish from other similarly pigmented Sebastes specimens. 

 We also use mitochondrial DNA (mtDNA) sequence 

 data to identify four putative pygmy rockfish specimens 

 representing a continuum of late-larval through pelagic 

 juvenile stages. The molecular results are used to con- 

 firm identifications based on morphological, meristic, 

 and pigmentation characters and to assure that the 

 assembled developmental series is monospecific. 



Methods 



Specimen collection 



Specimens of larval and pelagic juvenile pygmy rockfish 

 were obtained from research cruises conducted aboard 

 the NOAA RV David Starr Jordan off central California. 

 Specimens were collected in midwater (5-30 m) from 

 mid-May to mid-June, 1990-92, between Bodega Bay 

 (north of San Francisco) and Cypress Point (south of 

 Monterey Bay) by using a 26 mx26 m modified Cobb 

 midwater trawl (12.7-mm stretched-mesh codend liner). 

 Specimens also were collected during early March, 

 1992-93, between Salt Point (north of San Francisco) 

 and Cypress Point with a 5 mx5 m modified Isaacs-Kidd 

 (MIK) frame trawl with 2-mm net mesh and 0.505-mm 

 mesh codend. Specimens from the Cobb trawl were frozen 

 and specimens from the MIK frame trawl were preserved 

 in 95% ethanol for later analysis. 



Meristics, morphometries, and body pigmentation 



We examined pigmentation patterns and physical char- 

 acteristics of 122 pygmy rockfish larvae and pelagic 

 juveniles. Standard length (SL) was measured for each 

 individual and sizes ranged from 8.1 to 34.4 mm. Speci- 

 mens greater than 19.9 mm were identified by using 

 meristic characters (Chen. 1986; Matarese et al.. 1989; 

 Moreland and Reilly, 1991; and Laroche 1 ), and pigment 

 patterns were recorded. Specimens less than 20 mm 

 were identified initially from pigment patterns from a 

 series starting with the smallest (8.1 mm SL) identifi- 

 able individuals with complete fin-ray counts. Counts 

 of dorsal-, anal-, and pectoral-fin rays, and the number 

 of gill rakers on the first arch were recorded whenever 

 possible and subsequently used in identifications. Gill 

 raker counts were obtained only from fish larger than 



15 mm SL. 



We measured snout-to-anus length, head length, 

 snout length, eye diameter, body depth at the pectoral 

 fin base, body depth at anus, and pectoral-fin length on 



16 specimens ranging from 8.1 to 29.6 mm SL, follow- 

 ing Richardson and Laroche (1979). Head spination was 

 examined on thirty-three specimens (8.1 to 29.6 mm 



1 Laroche, W. A. 1987. Guide to larval and juvenile rock- 

 fishes (Sebastes) of North America. Unpubl. manuscript, 

 311 p. Box 216, Enosburg Falls, VT 05450. 



SL) that were stained with alizarin red-s. Terminol- 

 ogy for head spination follows Richardson and Laroche 

 (1979). In the following descriptions, larval and juvenile 

 lengths always refer to SL and pigmentation always 

 refers to melanin. 



Otolith examination 



Sagittal otoliths were removed from 61 larval and pelagic 

 juvenile pygmy rockfish (8.1-34.4 mm SL), and growth 

 increments were counted beginning at the first incre- 

 ment after the extrusion check (the mark in the otolith 

 formed when the larvae are released from their mother) 

 by using a compound microscope at lOOOx magnifica- 

 tion (see Laidig et al., 1991). No validation of the these 

 growth increments was performed during the present 

 study, and none has been conducted by other research- 

 ers. However, we assumed that these growth increment 

 counts corresponded to daily ages based on validation 

 of daily growth increments in other co-occurring rock- 

 fishes, namely shortbelly rockfish, S. jordani (Laidig et 

 al., 1991), black rockfish, S. melanops (Yoklavich and 

 Boehlert, 1987), bocaccio, S. paucispinis, chilipepper, 

 S. goodei, widow rockfish, S. entomelas, and yellowtail 

 rockfish, S. flavidus (Woodbury and Ralston, 1991). The 

 radius of the otolith was measured from the primordium 

 to the postrostral edge of the extrusion check for com- 

 parison with similar measurements from other Sebastes 

 spp. (as reported in Laidig and Ralston, 1995). Transfor- 

 mation from the larval stage to the pelagic juvenile stage 

 was ascertained by the presence of accessory primordia 

 (Laidig et al., 1991; Lee and Kim, 2000). 



Molecular confirmation 



Total genomic DNA was isolated from skeletal muscle 

 tissue of four larval and juvenile putative pygmy rock- 

 fish specimens by using a CTAB and phenol-chloro- 

 form-isoamyl alcohol protocol ( Winnepenninckx et al., 

 1993; Hillis et al., 1996). These four specimens ranged 

 in length from 15 to 27 mm and had pigment patterns 

 similar to the fish identified as pygmy rockfish in the 

 present tudy. Polymerase chain reaction (PCR) ampli- 

 fications and sequencing of partial mitochondrial DNA 

 regions (cytochrome b [cyt-6] and control region [CR]) 

 followed the methods of Rocha-Olivares et al. (1999a, 

 1999b). PCR products were verified on 29c agarose gels 

 and purified by using a QIAquick™ PCR Cleanup Kit 

 (Qiagen, Inc., Valencia, CA) following manufacturer 

 protocols. Complementary strand sequence data were 

 generated by using ABI PRISM' M DyeDeoxy™ termina- 

 tor cycle sequence chemistry on an automated sequencer 

 (Applied Biosystems, Model 377, Foster City, CA). 



Cytochrome b sequence data (750 base pairs) from 

 the four specimens were aligned with (previously gen- 

 erated) orthologous sequences from 119 individuals 

 representing 61 species of Sebastes (Rocha-Olivares et 

 al., 1999b). Species identifications, based on cyt-fo data, 

 were made by using distance-based cluster analyses in 

 PAUP v4.0b2 (Phylogenetic Analysis Using Parsimony, 



