Evolution and development of facial bone morphology in threespine sticklebacks

Charles B. Kimmel; Bonnie Ullmann; Charline Walker; Catherine Wilson; Mark Currey; Patrick C. Phillips; Michael A. Bell; John H. Postlethwait; William A. Cresko

doi:10.1073/pnas.0408533102

New Research In

Edited by David B. Wake, University of California, Berkeley, CA, and approved March 1, 2005 (received for review November 16, 2004)

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Fig. 1.
OP morphological variation among Alaskan threespine sticklebacks. (A) A left side view of the head (Alizarin Red staining without clearing). The OP is positioned on the hyoid arch, the most dorsal bone of a DV meristic series (10) that includes the scythe-shaped subopercle (SOP) and three sickle-shaped branchiostegal rays (BR). These bones function in opercular pumping. (B) Dissected, Alizarin Red-stained OPs from anadromous (Anchor River, Upper) and lake (Long Lake, Lower). The examples represent near-extreme high and low cases of variation along the VP axis (C). The sizes are shown as normalized to SL (scale bars, 2 mm). The lines marked JP and VP connect the landmarks J (joint), P (posterior), and V (ventral) that were readily assigned in every case by noting the locations of maximal curvature of the bone. We use the lengths of these lines in shape and size comparisons. (C) A morphospace showing the distribution of OP morphologies from all of the wild-captured Alaskan fish. VP_S and JP_S are SL-standardized lengths of VP and JP (B), made by taking the residuals of linear regressions of VP length and JP length on standard length (SL) for the lake data set. We excluded the anadromous fish from the regression equation, because the slope of the regression line for the lake fish alone is near 1.0 (Fig. 6). The upper data points are exclusively from anadromous fish (Anchor River, black; Rabbit Slough, gray). The lower data points are exclusively from lake fish (Boot Lake, dark blue; Mud Lake, light blue; Long Lake, orange; Bear Paw Lake, green; Bolo Lake, purple; Whale Lake, red). The theoretical diagonal line passes through the mean of the lake fish, and indicates variation in OP size without a change in shape.
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Fig. 2.
An early change in allometric development underlies OP evolution. Confocal microscope z stacks are shown as projections, from Alizarin Red-labeled, living, anesthetized larvae. Left side views, anterior to the left, are shown. (A) Detailed views at 7–11 DPF comparing OP morphologies of representative anadromous and lake larvae. The anadromous bones show relatively more prominent DV expansion. (B) Views including neighboring bones at 7–21 DPF in lake fish (the first three are the same individuals as in A). OP, opercle; SOP, subopercle; BR, branchiostegal ray. At the earliest stage, the OP is the only bone developing in the pharyngeal arches; then, other facial bones appear, including the other members of the OP-branchiostegal meristic series in the hyoid arch shown for the adult in Fig. 1 A. Like the OP, the other dermal bones begin development as lines, which then take on their specific shapes.
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Fig. 3.
Two phases of allometric OP growth differ between anadromous and lake fish. The data are from laboratory-reared fish derived from within-population crosses from Rabbit Slough (anadromous; black points), Bear Paw Lake, and Boot Lake (both lakes combined as red points; we observed no significant development differences between fish from the two lakes). Data were collected from 7 DPF through 260 DPF (anadromous) and through 136 DPF (lake). Statistical comparison (F ratio and P value for anadromous vs. lake) of slopes was performed by analysis of homogeneity of regression (55). (A) Linear–linear VP by JP plots (lengths in mm, not size-standardized). The data from both populations robustly fit straight lines; the slope is significantly higher for the anadromous fish. VP_anadromous= –0.23 + 1.72*JP_anadromous, r ²=0.99. SE = 0.0109. VP_lake= –0.24 + 1.48*JP_lake, r ²= 0.99. SE = 0.0200. Test of slopes: F _{1, 202}= 87.92; P < 0.0001. (B) An early, distinctive, and highly allometric growth phase shows up for both populations in log–log VP by JP plots of the same data. A larger slope means higher allometry (a slope of 1 means isometric growth). We fit two regression lines to each data set: “early period,” <12 DPF, the pair of lines to the left, and “late period,” >11 DPF, pair of lines to the right. Early period: log VP_anadromous= 2.31 + 2.66 *log JP_anadromous; r ²= 0.94; SE = 0.146. Lake: log VP_lake= 1.98 + 2.71 *log JP_lake; r ²= 0.96; SE = 0.217. Test of slopes: F _{1, 30}= 0.052; P = 0.82. Late period: log VP_anadromous= 0.32 + 1.25 *log JP_anadromous; r ²= 0.99; SE = 0.012. Lake: log VP_lake= 0.15 + 1.35 *log JP_lake; r ²= 0.99; S.E. = 0.012. Test of slopes: F _{1, 197}= 21.4; P < 0.0001. The difference in slopes is in the wrong direction to produce the smaller VP length of the adult lake fish by heterochronic acceleration.
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Fig. 4.
Between-population crosses reveal features of the genetic basis of OP morphology. Morphospace plots are shown as in Figs. 1 and 3. (A) Wild-captured parental populations, a subset of the data also replotted from Fig. 1C (Rabbit Slough, RS, gray; Boot Lake, Bt, red; Bear Paw Lake, BP, green). The means for these distributions of the parental fish OPs are shown by the colored circles in B–F.(B) Laboratory-reared F₁ progeny from a lake by lake (BP by Bt) cross. (C–F)F₁ (C and E) and F₂ (D and F) laboratory-reared progeny from two anadromous by lake crosses, RS by BP (C and D) and RS by Bt (E and F).
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Fig. 5.
An OP morphology major-effect locus maps to LG19. LOD score (blue) and percentage of the variance explained (pink) for the size-standardized VP component of OP variation plotted along the entire length of the linkage group are shown. Significant and highly significant marks were determined numerically by using permutation tests of the data across all linkage groups, as was the 95% confidence interval (yellow line) of the main peak by using bootstrap resampling. Data are from the RSxBP F₂ progeny shown in Fig. 4D. The presence of two peaks may mean that two QTLs contributing to OP ventral elongation are present on LG19, but also could be due to incorrect assignment of the stn185–187 marker positions. Further analyses will be required.

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Table 1. OP dimensions for the wild-captured fish

Group	n	Area_s	VP_s	JP_s
Anadromous	28	2.22 ± 0.42	1.53 ± 0.08	0.16 ± 0.04
Lake	96	0.00 ± 0.17	0.00 ± 0.04	0.00 ± 0.03
ANOVA, F _{1, 122}		400	392	8.68
P		<0.0001	<0.0001	0.004

Size standardizations as in Fig. 1. Data are expressed as means ± SE. See Table 2 for a more complete summary. ANOVA statistics, F ratios, and P values are comparisons between Anadromous and Lake fish.

Data supplements

Kimmel et al. 10.1073/pnas.0408533102.

Supporting Information

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Supporting Figure 6
Supporting Figure 7
Supporting Table 2
Supporting Table 3

Fig. 6. Opercles (OPs) vary in size but not dramatically in shape among fish from different lakes. (A) Plot of VP length versus JP length (not size-standardized) for the six lakes in the study set. Colors are as in Fig. 1. The points fit a straight line (r² = 0.89) with a slope of 1.42, indicating that the sizes of the OPs vary substantially whereas the relative dimensions of the bone remain constant (F_{1, 94} = 728; P < 0.0001). (B) Size-standardized VP_S versus JP_S plot shows a line (r² = 0.61) with a slope of 1.05. The value near 1 for the slope indicates that the two dimensions of the bone are co-varying independently of SL; i.e., the OP changes in size but not shape (F_{1, 94} = 144; P < 0.0001). Particular lakes can occupy different domains along the line (see also Table 2). For example, the normalized Bear Paw Lake OPs (green) are substantially larger than those from Mud Lake (light blue). (C) An overlay of two bones from Fig. 7, representing fairly extreme variation in normalized OP size, i.e., along the diagonal of the morphospace.

Fig. 7. Two axes of opercle (OP) shape variation. Pictorial representation of a subset of the OP morphology distribution shown in Fig. 1C, selected to show how shapes vary with reference to the two axes, VP and JP. The drawings are size standardized and color-coding is as in Fig. 1C. The upper inset shows where these selected examples fall on the morphospace. The lower inset shows an overlay of two bones where the principal variation is along the VP axis.

Table 2. Opercle dimensions for the wild captured populations

Group	n	SL (mm)	Area (mm²)	Area_S	T-K* (Area_S)	VP Length (mm)	VP_S	TK* (VP_S)	JP length, mm	JP_S	TK* (JP_S)
Anadromous^†
Anchor River	15	68.50 ± 0.97	26.66 ± 0.88	7.40 ± 0.65	A	7.59 ± 0.15	1.41 ± 0.11	A	4.49 ± 0.08	0.13 ± 0.06	A
Rabbit Slough	13	67.31 ± 0.56	26.65 ± 0.49	7.85 ± 0.52	A	7.75 ± 0.28	1.67 ± 0.09	A	4.48 ± 0.05	0.19 ± 0.05	A
Lake
Whale	21	44.89 ± 2.01	11.21 ± 1.10	1.11 ± 0.39	B	4.39 ± 0.23	0.18 ± 0.08	B	3.25 ± 0.16	0.15 ± 0.06	B
Bear Paw	15	46.39 ± 0.71	11.37 ± 0.43	0.69 ± 0.34	BC	4.47 ± 0.07	0.13 ± 0.05	B	3.36 ± 0.06	0.19 ± 0.05	B
Boot	15	51.23 ± 0.95	12.79 ± 0.55	0.23 ± 0.46	BC	4.88 ± 0.13	0.13 ± 0.12	B	3.45 ± 0.08	0.02 ± 0.07	BC
Bolo	15	39.05 ± 0.41	7.37 ± 0.19	-0.46 ± 0.17	CD	3.62 ± 0.05	-0.10 ± 0.04	BC	2.69 ± 0.04	-0.09 ± 0.03	CD
Long	15	54.40 ± 0.65	13.39 ± 0.48	-0.39 ± 0.36	CD	4.94 ± 0.09	-0.07 ± 0.07	BC	3.51 ± 0.05	-0.10 ± 0.04	CD
Mud	15	55.57 ± 0.71	12.61 ± 0.37	-1.63 ± 0.16	D	4.76 ± 0.07	-0.35 ± 0.05	C	3.44 ± 0.05	-0.23 ± 0.03	D

Size standardizations (Area_S, VP_S, and JP_S) are as in Fig. 1. The columns show means ± SE.

*Tukey-Kramer comparisons. Levels not connected by the same letter are significantly different (P < 0.05).

^†Anchor River vs. Rabbit Slough comparisons. Area_S : F_1,26 = 0.280; P = 0.60. VP_S: F_1,26 = 3.13; P = 0.09. JP_S: F_1,36 = 0.443; P = 0.51

Table 3. Opercle (OP) dimensions for the genetic crosses

Hybrids	n	Generation	VP_S mean ± SE	Variance of VP_S	JP_S mean ± SE	Variance of JP_S
(Lake ´ Lake)
Whale ´ Boot	32	F₁	0.22 ± 0.05	0.086	0.29 ± 0.04	0.041
Bear Paw ´ Boot	50	F₁	1.45 ± 0.05	0.125	1.16 ± 0.05	0.102
(Anadromous ´ Lake)
Rabbit S. ´ Bear Paw	49	F₁	0.54 ± 0.04	0.082	0.03 ± 0.02	0.021
Rabbit S. ´ Bear Paw	99	F₂	1.03 ± 0.04	0.141	0.46 ± 0.03	0.065
Rabbit S. ´ Whale	25	F₁	0.62 ± 0.04	0.048	0.01 ± 0.03	0.020
Rabbit S. ´ Whale	41	F₂	1.36 ± 0.07	0.229	0.40 ± 0.04	0.057
Rabbit S. ´ Boot	25	F₁	0.87 ± 0.05	0.055	0.26 ± 0.03	0.025
Rabbit S. ´ Boot	52	F₂	1.21 ± 0.06	0.211	0.69 ± 0.04	0.093