Handed behavior in hagfish-an ancient vertebrate lineage and a survey of lateralized behaviors in other invertebrate chordates and elongate vertebrates.
Vertebrates exhibit a surprising variety of handed behaviors from left-footed food grasping by parrots (Harris, 1989; Rogers and Workman, 1993) to right-pawed face wiping by toads (Naitoh and Wassersug, 1996). However, uncertainties remain about how widespread individual or population level handed behaviors are across the more basal lincages of vertebrates. Rooted between jawed vertebrates and non-vertebrate chordates along with lampreys (Fig. I; Heimberg et al., 2010), hagfish (Myxinoidea) are peculiar eel-like, boneless, jawless, and sightless fish that exhibit several unique behaviors including a conspicuously asym-metrical one. They produce copious amounts of slime to deter predators (Strahan, 1963; Martini, 1998; Zintzen et al., 2011). They form a traveling knot" to escape a grip, to get them rid of their own slime, or to anchor the body when ripping flesh while scavenging (Adam, 1960). And, at least in one dominant genus (Eptatretus), the body forms a tight coil when at rest (Strahan, 1963; Martini, 1998) (Fig. 2A). A deep origin of the lineage, its primitive appearance, and these other unique behaviors make hagfish an interesting model for studying lateralized behavior in the earliest vertebrates.
Both clockwise and counterclockwise coiling occurs fre-quently in Eptatretus, a genus of hagfish that contains more than 70% of the approximately 70 known hagfish species (Fernholm, 1998; Kuo et al., 2003). Previous accounts of hagfish behaviors casually mention that healthy individuals of Eptatretus coil at rest, both in the field and in captivity, unless burrowing in soft sediment or confined in a narrow space (Strahan, 1963; Martini, 1998). In the first systematic treatment of this behavior, we use the northeastern Pacific inshore hagfish Eptatretus stoutii (Lockington, 1878) to test whether (1) individual E. stoutii exhibit a preferred direction of coiling, and (2) the population exhibits an overall left or right bias in this behavior. We also present a synthesis of coiling and other lateralized behaviors from a broad sample of other elongate vertebrates and non-vertebrate chordates. Do lateralized behaviors occur in other elongate vertebrates and non-vertebrate chordates? Are coiling or other lateral-ized behaviors functionally related or phylogenetically linked? Answers to these questions test whether or not lateralized behaviors in non-vertebrate chordates and verte-brates share a common evolutionary origin.
Materials and Methods
We trapped 40 hagfish (Eptatretus stoutii) from a depth of about 80 m in Barkley Sound, British Columbia, Canada (48[degrees]84'96.37"N; 125[degrees]13' I 8.01"W). Each individual was held separately in a 100-liter tank with running seawater (12-15[degrees] C) and a dark cover for 2 to 3 months and monitored as frequently as every 10 to 30 min for 10 to 12 h per day at the Bamfield Marine Sciences Centre. Coiling direction was recorded until 50 observations were obtained per individual. Coiling direction was determined moving from the tail to the head as viewed from the dorsal side (Fig. 1A). The head was always to the outside of the spiral regardless of coiling direction and regardless of whether individuals were resting on their ventral or dorsal side. We logged a new observation only after confirming, either by direct observation of movement or by the animal's change of posture or location in the tank, that a hagfish had uncoiled from its previous resting position. Individuals sometimes responded to the disturbance of opening the tank cover by uncoiling (scored for direct observation of movement), or sometimes they did not respond at all. Hagfish are generally quite sedentary and rest in the same position and location in the aquarium over a week in some cases (TM, pers. obs.).
We tested whether coiling direction in each individual occurred in one direction more often than expected due to chance by comparing observed frequencies to critical values for a test of equal proportions. For each individual hagfish, we calculated a handedness score h:
h = [n.sub.c] - [n.sub.cc](1)
Where [n.sub.c] is the number of clockwise coiling and [n.sub.cc] is the number of counterclockwise coiling events. The frequency distribution of h reveals modes of asymmetry (Palmer,2004). If individual hagfish do not have a preferred coiling direction, the distribution of h will be unimodal with a mean near zero. If individuals have a preferred direction, and if preferred directions are not heritable, the distribution will be symmetrically bimodal with a mean near zero. If individuals have a preferred direction, and if the degree of side dominance is heritable, the distribution could take many shapes. but the mean should depart from zero.
We also tested whether individual hagfish repeated an immediately preceding coiling direction more than expected from the observed frequencies of clockwise or counter-clockwise coiling for that individual, regardless of preferred orientation. We calculated z scores for repeating the preced-ing coiling direction for each individual, using the standard formula for z test statistics for test of proportion (calculated separately for clockwise and counterclockwise coiling):
[MATHEMATICAL NOT REPPODUCIBLE IN ASCII] (2)
Where [R.sub.i] is the frequency of events in which an individual i repeated the same coiling direction as the immediately preceding observation, where [f.sub.i] is the overall frequency of that coiling orientation, and where [p.sub.i] is a proportion of that direction in 50 observations. A square of [p.sub.i] estimates the probability of the same coiling direction occurring twice consecutively due to chance based on a proportion of that direction in a full sample of 50 observations for that individual. The first observation of each coiling orientation was excluded because the preceding coiling direction was un-known. To evaluate whether hagfish repeat the same coiling orientation at the population level more often than expected due to chance, we calculated [z.sub.t], for the entire data set for each coiling direction by substituting each term in Equation 2 with the sum of all individuals.
To rule out the possibility that individual hagfish developed a preference during the experiment, we performed paired Wilcoxon signed rank tests on both the strength of handedness [absolute value of h] and the frequency of repeated coiling orientation [R.sub.t] in the data set of all individuals between the first 10 observations and the last 10 observations. We also calculated Spearman's rank correlation coefficient ([r.sub.s]) with body length for these two variables to test whether body size affected individual handedness or repeatability of coiling direction.
Out of 40 individual hagfish, 29 exhibited significantly handed coiling at a = 0.05. However, all but one individual coiled in one direction (clockwise [C] or counterclockwise [CC]) 30 or more times in the same direction out of 50 observations (Table 1; h > 10 or h < -10). Only three individuals departed from the critical values by a count of two or more. The ratio between statistically significant C coilers and CC coilers (16:13) did not differ significantly from equal odds (50:50) (z test for equal proportions; P = 0.71), and the frequency distribution of individual handed-ness scores was clearly bimodal with a mean near zero (Fig. 2B). No individual fell in the interval around equal odds (h = 0). Within the clearly identified C and CC coilers, about half of the individuals in each group coiled in that direction 15 to 25 times more than in the other direction. Both C and CC coilers have long tails of frequency distribution away from even odds, and four individuals coiled almost exclu-sively in one direction (40 or more times out of 50). Among sequential observations, 30 of 40 individuals repeated the immediately preceding coiling direction more often than expected from the observed frequencies of C and. CC coil-ing, regardless of their overall preferred directions, and four more individuals did so in one of the coiling directions (Table 1). In the entire data set, z, scores of repeated coiling calculated for each coiling direction (C: n = 40; df = 39; [z.sub.sC] = 30.47; P < 0.01; CC: n = 40; df = 39; [z.sub.sCC] = 29.93; P < 0.01) were highly significant. Therefore, most individ-uals repeated the same coiling directions more often than expected due to chance at the population level.
Coiling statistics for individual hagfish used in this study, ordered by increasing body length (BL) in mm Specimen BL [n.sub.c] [n.sub.cc] [n.sub.rc]c 1 260 44 6 39 2 294 32 18 ([section] 26 ) 3 302 14 36 4 4 317 35 15 27 5 319 21 29 ([section] 8 [section] ) 6 322 31 19 ([section] 21 ) [section] ) 7 324 14 36 5 8 328 20 30 ([section] 9 [section] ) 9 345 17 33 10 10 348 12 38 4 11 350 19 31 ([section] 13 [section] ) 12 366 17 33 9 13 367 31 19 ([section] 22 ) [section] ) 14 368 7 43 5 15 371 19 31 7 16 380 35 15 26 17 386 37 13 31 18 393 15 35 7 19 419 31 19 ([section] 18 [section] ) 20 419 37 13 26 21 421 12 38 4 22 433 35 15 26 23* 446 10 40 1 24 448 34 16 21 25 452 36 14 29 26 465 5 45 0 27* 470 32 18 ([section] 16 ) 28 472 37 13 27 29 477 15 35 8 30 480 36 14 29 31 482 15 35 6 32 493 13 37 3 33 498 36 14 27 34* 502 17 33 5 35 508 15 35 4 36* 514 14 36 3 37* 523 35 15 23 38 576 20 30 ([section] 10 [section] ) 39 586 48 2 45 40 698 18 32 ([section] 10 ) Specimen [n.sub.rcc] 1 1 2 12 3 26 4 7 5 17 6 9 7 27 8 19 9 27 10 30 11 24 12 25 13 11 14 40 15 21 16 6 17 8 18 27 19 7 20 ([section] 3 ) 21 29 22 7 23* ([section] 30 ([section] ) ) 24 ([section] 4 ([section] ) ) 25 8 26 OD 40 ([section] ) 27* ([section] 3 ([section] [section] ) ) 28 4 29 28 30 6 31 26 32 26 ([section] ) 33 5 34* 20 ([section] ) 35 24 36* (0 24 ([section] ) 37* 3 ([section] [section] ) 38 19 39 ([section] 0 ([section] [section] [section] ) ) 40 24 * Gravid females. Statistical abbreviations: [n.sub.c], number of clockwise events; [n.sub.cc], number of counterclockwise events ([H.sub.o]: [n.sub.c] = [n.sub.cc]; critical values from Table Q; Rohlf and Sokal 1995); [n.sub.rC], number of clockwise events that were preceded by a clockwise coiling orientation (tested for z score with [H.sub.o] : [p.sub.rc] = [p.sub.c.sup.2]; see Methods); [n.sub.rcc], number of counterclockwise events that were preceded by a counterclockwise coiling orientation (tested for z score with [H.sub.o]: [p.sub.rcc] = [p.sub.cc.sup.2]; see Methods). Symbols in parentheses indicate level of significance ([section] failure to reject [H.sub.o] at [alpha] = 0.01; [section]: failure to reject H0 at a = 0.05). Appendix presents the complete table of statistical tests.
Individual hagfish did not reinforce their coiling orienta-tion during the study. The paired Wilcoxon signed rank test between the first 10 and last 10 observations revealed no significant difference either in the strength of handedness ([absolute value of h] n = 40; df = 39; WS [sum of Wilcoxon's signed ranks] = 222.5; [alpha] = 0.01) or in the frequency of repeating the same coiling orientation twice consecutively ([R.sub.t]: n = 40; df = 39; WS = 457.5; [alpha] = 0.01). No significant correlation was observed between body size and the strength of handed behavior ([absolute value of h] n = 40; df = 39; [r.sub.s] = 0.096; T = 0.59; p = 0.55) or the frequency of repeated coiling in the same direction ([R.sub.t]: n = 40; df = 39; [r.sub.s] = -0.304; T = 1.97; p = 0.056) even though body lengths ranged from 260 to 698 mm.
Coiling behavior of individual hagfish is handed
Individual hagfish (Eptatretus stoutii) clearly exhibited handed coiling behavior in a laboratory setting. The majority (29 of 40 individuals) showed a statistically significant preference toward one coiling direction. The bimodal dis-tribution of individual handedness scores (Fig. 2B) indicates that preferred coiling orientation varied at random among individuals, which implies no genetic determination to preferred direction (Palmer, 2004, 2005). Frequency-dependent selection has been argued to maintain roughly equal fre-quencies of right and left bending mouth morphs in one animal example: scale-eating cichlid fish (Hori, 1993). However, doubts have been raised about whether direction of mouth bending is actually inherited in this species, and increasing evidence suggests that it may be induced via developmental plasticity in response to strongly lateralized behavior (Palmer, 2010; Kusche et al., 2012; Lee et al., 2012).
For hagfish, coiling is a stable resting posture that may help them avoid detection by predators or reduce drag. But we see no obvious advantage to coiling in a particular direction. We have also not found any anatomical correlates of coiling orientation. Even the right-biased gonads appear not to bias coiling orientation, likely because they are suspended near the midline in the visceral cavity (Marinelli and Strenger, 1956). Among five gravid females we observed (noted with an asterisk [*] in Table 1), the ratio of significant C to CC coilers is 2:3 at [alpha] = 0.05 and 1:3 at [alpha] = 0.0 I. So both C and CC coilers occurred among gravid females bearing large gonads. The number of gill pouches occasionally differs between the right and left sides (Martini and Beulig, 2013), but such asymmetry is rare. None of more than 20 Eptatretus specimens we dissected exhibited any gill asymmetry, and no consistent bias toward one side appears to exist in cases reported in the literature.
The handed coiling behavior of individual Eptatretus is most easily explained as a reinforced behavior following a random initial choice, as observed in paw use by mice (Ribeiro et al., 2011). Indeed, current coiling direction appears to strongly bias subsequent choice of coiling direc-tion in hagfish. Even though no significant relationship existed between body size and either the strength of hand-edness or the tendency to repeat the same coiling orienta-tion, young E. stoutii clearly developed preferred coiling directions at or before the smallest sizes we examined (body length of 260 mm).
Coiling and knotting behavior in hagfish
Although many species of the dominant hagfish genus Eptatretus coil, coiling does not appear to occur in one well-studied species of the other common genus, Myxine glutinosa (Strahan, 1963). So, either coiling behavior evolved once in Eptatretus, or the lack of coiling behavior in Myxine is a secondary loss. Although neither lineage is paraphyletic relative to the other (Fernholm, 1998; Kuo et al.. 2003, 2010; Chen et al., 2005; Fernholm et al., 2013), Eptatretus is generally believed to retain more plesiomor-phic morphological features relative to the specialized bur-rower Myxine (Strahan, 1963; Martini, 1998; Miyashita, 2012), so the lack of coiling in Myxine may be derived. A recent molecular phylogenetic analysis resolved two previ-ously known species of Eptatretus into a newly designated genus Rubicundus as a sister group to eptatretines and myxinines (Fernholm et al., 2013). Tests for coiling behav-ior in Rubicundus and other species of Eptatretus would therefore resolve whether coiling behavior was an ancestral state in hagfish.
Species of both Eptatreutus and Myxine exhibit another conspicuously asymmetric behavior. They tie themselves in-and slip through-a knot to escape from attack, to rid themselves of their mucous secretions and other debris on the body, and in macrophagous feeding (Adam, 1960; Stra-han, 1963; Martini, 1998; Zintzen et al., 2011). Such knots also come in right-handed (the body crosses over the head before the tail passes through the loop) and left-handed forms (the body crosses under the head before the tail passes through the loop) from either head or tail. We did observe this behavior occasionally, but not frequently enough for us to test for concordance with coiling orientation. Nonetheless, it would be interesting to know whether either Myxine or Eptatretus species exhibit consistent handed behavior in knotting and whether, in Eptatretus, chirality of knotting correlates with chirality of coiling.
Hagfish coiling behavior is distinct from those of other vertebrates
Coiling behaviors occur in several elongate vertebrates (Table 2). Some notable examples are the following:
Taxa and behavior Ind. Pop. Pref. Life Sex stage Hemichordata Enteropneust (various abyssal taxa); - - - Adult - spiral fecal trail Enteropneust Yes Yes C Adult - (Glandiceps hacksi); spiral swimming Cephalochordata Lancelets (Branchiostoma); Yes Yes C Late - spiral swimming larva Lancelets (Branchiostoma); resting on side No No N/A Adult - Tunicata Ascidian tadpoles; Yes Yes C Larva - spiral swimming Myxinoidea Hagfish Yes No C, CC Adult Both (Eptatretus); coiling at rest Hagfish -- -- -- Adult Both (Eptatretus, Myxine): traveling knot Petromyzontiformes Lamprey No No N/A Larva -- (Petromyzon); resting on side Lamprey -- -- -- Adult M (Petromyzon); males wrapping around females Gnathosiomata Sturgeon Yes No C, CC Juvenile -- (Acipenser); rotational swimming Eel (chlopsids, -- -- -- Larva -- congrids, and muraenids); leptocephalus curling Pricklebacks & -- -- -- Adult M gunnels (stichaeids & pholids); curling around egg mass Catfish (silurids) -- -- -- Adult M and loaches (cobitids); males enfolding females Wolffish -- -- -- Adult M (Anarichas); male rolls over to side and bends in U-shape Lungfish (Protopterus); curling within aestivation burrow -- -- -- Adult Both amphibians -- -- -- Adult Both (salamanders. caecilians, and lysorophoids); curling for various purposes Taxa and behavior Head Coil pos. form Hemichordata Enteropneust (various abyssal taxa); spiral Out Tight: fecal trail multiple Enteropneust (Glandiceps N/A Loose; hacksi); spiral swimming multiple Cephalochordata Lancelets (Branchiostoma); spiral N/A N/A swimming Lancelets (Branchiostoma); resting on side N/A N/A Tunicata Ascidian tadpoles; spiral N/A N/A swimming Myxinoidea Hagfish (Eptatretus); Out Tight; coiling at rest multiple Hagfish (Eptatretus, N/A Tight; Myxine): traveling knot single Petromyzontiformes Lamprey (Petromyzon); N/A N/A resting on side Lamprey (Petromyzon); Out Loose; males wrapping around multiple females Gnathosiomata Sturgeon (Acipenser); N/A N/A rotational swimming Eel (chlopsids, congrids, In Loose: and muraenids); multiple leptocephalus curling Pricklebacks & gunnels Out Loose: (stichaeids & pholids); single curling around egg mass Catfish (silurids) and Out Loose; loaches (cobitids); males single enfolding females Wolffish (Anarichas); male Out Loose; rolls over to side and single bends in U-shape Lungfish (Protopterus); curling within aestivation burrow Out Loose; single amphibians (salamanders. In. Loose; caecilians, and out single, lysorophoids); curling for multiple various purposes Taxa and behavior Setting Function Seasonality Ref# Hemichordata Enteropneust (various abyssal taxa); Aquatic; Feeding Perennial 1 spiral fecal trail substrate Enteropneust Aquatic; Dispersal Seasonal? 2 (Glandiceps suspension hacksi); spiral swimming Cephalochordata Lancelets (Branchiostoma); Aquatic; Dispersal Perennial 3 spiral swimming suspension Lancelets (Branchiostoma); resting on side Aquatic; Resting; Perennial 3 substrate defense? Tunicata Ascidian tadpoles; Aquatic; Dispersal Perennial 3 spiral swimming suspension Myxinoidea Hagfish Aquatic; Resting; Perennial 4 (Eptatretus); substrate defense? coiling at rest Hagfish Aquatic Defense; Perennial 5 (Eptatretus, feeding Myxine): traveling knot Petromyzontiformes Lamprey Aquatic; Resting; Perennial 4 (Petromyzon); substrate defense? resting on side Lamprey Aquatic; Mating Seasonal 6 (Petromyzon); suspension males wrapping around females Gnathosiomata Sturgeon Aquatic; Locomotion 7 (Acipenser); suspension Perennial rotational swimming Eel (chlopsids, Aquatic; Defense Perennial 8 congrids, and suspension muraenids); leptocephalus curling Pricklebacks & Aquatic; Parental Seasonal 9 gunnels (stichaeids substrate care & pholids); curling around egg mass Catfish (silurids) Aquatic; Maring Seasonal 10 and loaches suspension (cobitids); males enfolding females Wolffish Aquatic; Courtship Seasonal I1 (Anarichas); male substrate rolls over to side and bends in U-shape Lungfish (Protopterus); curling within aestivation burrow Terrestrial; Resting Seasonal 12 burrow amphibians Terrestrial; Resting; Perennial, 13 (salamanders. substrate; defense; seasonal caecilians, and burrow parental lysorophoids); care curling for various purposes Two different categories of behaviors are listed under the higher taxonomic headings: coiling or curling behaviors in chordates: potentially lateralized behaviors in outgroups of vertebrates. Behaviors are compared in different categories: mode of asymmetry, life history, morphology, and ecological contexts. Tests of individual preference and population bias indicate whether handedness develops at the individual or population level. Dash (-) indicates no information. N/A = not applicable. *Asymmetry mode. Ind: individuals exhibit a bias towards one side?; Pop: population bias toward one side?; Pref: preferred orientation? (C-clockwise, CC-counterclockwise). *Life history. Life stage: life stage that exhibits the behavior (larva, late larva, adult); Sex: (M-male only, both-evident in both sexes). [section] Morphology. Head pos: location of head in coiled position? (out-outside of coil, in-inside of coil); Coil form: tightness of coil (tight-body wall in contact between revolutions, loose-body wall not in contact between revolutions), number of revolutions (single, multiple). Ecological context. Setting: where coiling behavior is observed (aquatic vs. terrestrial; suspension-in the water column, substrate-on the surface of substratum, burrow-in below-ground burrows); Function: the possible adaptive significance of the behavior; Seasonality: does behavior occur year-round (perennial) or only during certain seasons (seasonal)? # Ref. Source of observations: 1) Smith et al. (2005), Anderson et al. (2011), 2) Urata et al. (2012), 3) Gislern (1930), 4) this study, 5) Adam (1960), Zintzen etal. (2011), 6) Lotion et al. (2000), 7) Izvekov et al. (2014), 8) Miller (2009), Miller et al. (2013), 9) Qasim (1957), Hughes (1986), 10) Maehata (2002), Bohlen (2008), 11) Johannessen et al. (1993), 12) Greenwood (1986), 13) Trauth et al. (2006), Olson (1971), 14) Roth (2003), Heatwole er al. (2007).
* Male lampreys wrap around females during spawning (Beamish and Neville, 1992; Lorion et al., 2000).
* Leptocephalus larvae of three families of eels (Chlop-sidae, Congridae, and Muraenidae) coil when sus-pended in water, and at least the latter two families exhibit both clockwise and counterclockwise coiling in published figures (Miller, 2009; Miller et al., 2013).
* Stichaeid and pholid perciforms (pricklebacks and gunnels) guard their egg masses by curling around them (Qasim, 1957; Hughes, 1986; Coleman, 1992).
* Silurid and cobitid males (catfishes and loaches) coil around conspecific females during spawning (Maehata, 2002; Bohlen, 2008).
* Males of wolffish (Anarhichas) roll over to one side and bend the body in inverted U shape prior to mating (Johannessen et al., 1993).
* Lungfish curl from the tail first with the snout pointing upward in estivation burrows (Johnels and Svennson, 1954; Greenwood, 1986).
* Lysorophoids (an extinct family of elongate lepospon-dyl tetrapods) are often found coiled within burrows or nodules (Wellstead, 1991). At least one specimen (UCLA VP 2801; Olson, 1971) is coiled clockwise; a reconstruction based on several specimens (Olson and Bolles, 1975) also exhibits clockwise coiling.
* Elongate amphibians such as salamanders and caecil-ians coil under many conditions including resting, brooding eggs, defense, and estivation (Cochran, 1911; Heatwole, 1960; Brodie, 1977; Brodie et al., 1984; Trauth et al., 2006; Fontenot and Lutterschmidt, 2011).
* Elongate squamates, especially snakes, coil for many functional purposes including resting, feeding, defense, estivation, and hibernation (Roth, 2003; Heatwole et al., 2007).
Although these behaviors are all described as coiling or urling, a comparative analysis reveals many differences .mong them and from the coiling behavior of hagfish (Table ;). These differences concern modes of asymmetries, life-History traits, morphology of coiling, and ecological con-exts. Unfortunately, for none of these behaviors do we now whether (a) individuals have a preferred orientation; or (b) a population bias exists. Either they have not been tudied systematically, or the presence of handedness is nconclusive. For example, 3 out of 30 individuals of cot-onmouth snakes coiled clockwise more frequently than ounterclockwise (P < 0.05; Roth, 2003). However, a fol-Dw-up experiment revealed no such preference in the same axon or in the closely related copperhead snakes (Heatwole 't al., 2007). In some cases, sample size appears sufficiently arge for a statistical test but quantitative data were not eported (e.g., for the lysorophoid Brachydectes, a single axiality has yielded more than 40 nodules, each likely ontaining a coiled skeleton; Hembree et al., 2005).
Clearly, such coiling behaviors warrant systematic study n these other animals. Nonetheless, the available evidence uggests that Eptatretus coiling is unique for having the Lead outside the spiral, showing strong individual prefer-nce for either direction, and using it as a primary resting losture in contact with the substrate, regardless of sex, eason, or life stage.
Asymmetric behaviors in basal vertebrates and their relatives likely have independent origins
Even if hagfish coiling is unique in detail, the mere presence of lateralized behavior may be informative. Is any lateralized behavior likely to have existed in the last common ancestor of living vertebrates? This question was previously considered in the context of various hypotheses that incorporated bilaterally asymmetrical fossil echinoderms in chordate evolution (Gislen, 1930; Jefferies, 1986; Gee, 1996). However, none of the authors addressed whether or not any lateralized behaviors in non-vertebrate chordates could be compared with those in the living vertebrates.
Besides hagfish, some basal vertebrates and vertebrate relatives exhibit asymmetrical resting postures. Both am-mocoete larvae of lampreys and the non-vertebrate chordate lancelets (Cephalochordata) rest on one side of the body. No marked difference exists between the frequencies of right and left sides at the individual level (ammocoetes: 41 to 45 observations each for six specimens, with 134 total right-side down, 125 total left; lancelet adults: 18 to 31 observations each for four individuals, with 42 right and 59 left; Gislen, 1930). Deep-sea acorn worms (Hemichordata) leave spiral fecal trails on the sea floor while feeding, and these are clockwise or counterclockwise with the head oriented out as in hagfish (Smith et al., 2005; Anderson et al., 2011). However, no consecutive observations were made to determine preferred orientation at the individual or population level.
Swimming does appear to be lateralized in some vertebrate relatives. Lancelet larvae switch from cilia-driven counterclockwise spiral swimming to muscle-driven clockwise spiraling after their peculiarly asymmetric metamorphosis, whereas ascidian tadpole larvae (Tunicata) consistently swim in a clockwise spiral (Gislen, 1930). Even some benthic acorn worms rotate clockwise while swimming (Urata et al., 2012). Juvenile sterlet sturgeons (Acipenser ruthenus) have either clockwise or counterclockwise bias in rotational swimming at the individual level, but no statistically significant bias was observed at the population level (Izvekov et al., 2014).
However, these swimming behaviors have a different morphological basis. In lancelet adults, the preoral hood extends anteriorly from the right metapleural fold, and the right series of somites develop half a segment posterior to the left series (Schubert et al., 2001). Removal of the hood leads to the loss of spiral swimming (Gislen, 1930). Among tunicates, Grave (1926) attributed the clockwise spiral swimming of ascidian larvae to oblique contractile fibrillae within the tail muscles. The tail undulates predominantly toward the left in Distaplia occidentalis (Berri11, 1950; McHenry, 2001). Such torsion and yawing may be augmented by a bilaterally uneven mass distribution and the lack of a bilateral sensory circuit that would facilitate corrective torque against yawing (McHenry and Strother, 2003; McHenry, 2005). There is no information on what drives clockwise swimming in acorn worms. As such, these curious behaviors cannot predict ancestral condition for vertebrates, and they have no bearing on the origin of coiling behavior in hagfish.
Given the information outlined so far, neither coiling behaviors nor lateralized behaviors are readily comparable between chordate lineages. Neither are they phylogeneti-cally congruent in the currently accepted tree (summarized in Fig. 1). That is, it takes a smaller number of changes to assume independent origins of the behaviors than to assume that a behavior arose in a common ancestor and was variably modified in some lineages and lost in others. Taken together, these observations lead to the following conclusions. First, the coiling behavior of Eptatretus has no apparent parallel among non-vertebrate chordates, and various coiling behaviors in vertebrates almost certainly evolved independently in each lineage. Second, individual preferences in coiling directions of Eptatretus likely develop via repetition of previous coiling directions, but arc not biased at the population level, analogous to the development of paw preference in mice (Ribeiro et al., 201 I ) and the individual bias in direction of rotational swimming in sturgeons (Izvekov et al., 2014). So, whereas coiling itself may be advantageous in some way, direction of coiling appears not to matter. Finally, no known lateralized behavior in living chordates can be inferred to have been present in the ancestral vertebrate.
This research was supported by scholarships from the Alberta Ingenuity Fund, Bamfield Marine Sciences Centre, and Vanier CGS to T.M. and NSERC Discovery Grant (A7245) to A.R.P., and approved under the animal use policy at BMSC and the University of Alberta. We thank B. Anholt, P. Currie, K. Gale. G. Goss. E. Koppelhus, K. Miyashita, E. Montgomery, J. Pierce, and D. Riddell for logistical support. T.M. thanks the faculty, students, and assistants for Embryology 2013-especially A. Accorsi, R. Behringer, A. Edgar, J. Henry, L. Maya Ramos, L. Linden, J. Park, and G. Smoke-Vaughan-for six weeks of scientific adventures at the Marine Biological Laboratory. This paper is a tribute to one of the themes during the course: linking natural historical observations to yield an evolutionary insight.
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List of specimens (BL, body length in mm) with statistics used in this paper Specimen BL [n.sub.c] [n.sub.cc] h [n.sub.rc] [n.sub.rcc] 1 260 44 6 38 39 1 2 294 32 18 14 26 12 3 302 14 36 -22 4 26 4 317 35 15 20 27 7 5 319 21 29 -8 8 17 6 322 31 19 12 21 9 7 324 14 36 - /1 5 27 8 328 20 30 - 10 9 19 9 345 17 33 - 16 +10 27 10 348 12 38 - 26 4 30 11 350 19 31 -1.2 13 24 12 366 17 33 -16 9 25 13 367 31 19 12 22 II 14 368 7 43 -36 5 40 15 371 19 31 - 12 7 21 16 380 35 15 20 26 6 17 386 37 13 24 31 8 18 393 15 35 -20 7 27 19 419 31 19 12 18 7 20 419 37 13 24 26 3 21 421 12 38 -26 4 29 22 433 35 15 20 26 7 23* 446 10 40 -30 I 30 24 448 34 16 18 21 4 25 452 36 14 22 29 8 26 465 5 45 -40 0 40 27* 470 32 18 14 16 3 28 472 37 13 24 27 4 29 477 15 35 -20 8 28 30 480 36 14 22 29 6 31 482 15 35 -20 6 26 32 493 13 37 -24 3 26 33 498 36 14 22 27 5 34* 502 17 33 -16 5 20 35 508 15 35 -20 4 24 36* 514 14 36 -22 3 24 37* 523 35 15 20 23 3 38 576 20 30 -10 10 19 39 586 48 2 46 45 0 40 698 18 32 -14 10 24 Specimen [P.sub.c] [P.sub.cc] [Z.sub.rc] [Z.sub.rcc] 1 0.88 0.12 2.08 3.48 2 0.64 0.36 4.86 7.07 3 0.28 0.72 3.08 2.66 4 0.70 0.30 3.55 5.36 5 0.42 0.58 2.62 3.03 6 0.62 0.38 3.55 4.29 7 0.28 0.72 4.11 3.00 8 0.40 0.60 3.73 3.31 9 0.34 0.66 6.37 4.66 10 0.24 0.76 4.36 2.87 11 0.38 0.62 6.97 4.68 12 0.34 0.66 5.59 3.94 13 0.62 0.38 3.93 5.63 14 0.14 0.86 14.38 3.14 15 0.38 0.62 2.95 3.55 16 0.70 0.30 3.20 4.43 17 0.74 0.26 3.78 8.27 18 0.30 0.70 5.36 3.55 19 0.62 0.38 2.43 2.95 20 0.74 0.26 2.11 2.52 21 0.24 0.76 4.36 2.54 22 0.70 0.30 3.20 5.36 23* 0.20 0.80 1.09 1.68 24 0.68 0.32 2.00 2.10 25 0.72 0.28 3.67 7.20 26 0.10 0.90 -0.20 1.68 27* 0.64 0.36 1.21 0.58 28 0.74 0.26 2.44 3.67 29 0.30 0.70 6.29 3.89 30 0.72 0.28 3.67 5.14 31 0.30 0.70 4.43 3.20 32 0.26 0.74 2.52 2.11 33 0.72 0.28 3.00 4.11 34* 0.34 0.66 2.46 2.16 35 0.30 0.70 2.56 2.52 36* 0.28 0.72 2.04 1.98 37* 0.70 0.30 2.18 1.62 38 0.40 0.60 4.36 3.31 39 0.96 0.04 0.91 -0.04 40 0.36 0.64 5.63 4.13 Abbreviations as in text. [n.sub.c], number of clockwise events; [n.sub.cc], number of counterclockwise events; h, handedness score ([n.sub.c] - [n.sub.cc); number of clockwise events that were preceded by a clockwise coiling orientation; [n.sub.rcc], number of counterclockwise events that were preceded by a counterclockwise coiling orientation; [P.sub.c] proportion of clockwise events in 50 observations; [P.sub.cc], proportion of counterclockwise events in 50 observations; [z.sub.rc], z score for number of clockwise events that were preceded by a clockwise coiling orientation for test of proportion ([P.sub.rc] = [P.sub.[c.sup.2]]); [z.sub.rcc], z score for number of counterclockwise events that were preceded by a counterclockwise coiling orientation for test of proportion ([P.sub.rcc] = [P.sub.c[c.sup.2]]). Asterisks (*) indicate gravid females.
TETSUTO MIYASHIITA* AND A. RICHARD PALMER
Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada ThG 2E9
Received 16 August 2013; accepted 18 February 2014.
* To whom correspondence should be addressed. E-mail: firstname.lastname@example.org
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|Author:||Miyashiita, Tetsuto; Palmer, A. Richard|
|Publication:||The Biological Bulletin|
|Date:||Apr 1, 2014|
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