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Handed behavior in hagfish-an ancient vertebrate lineage and a survey of lateralized behaviors in other invertebrate chordates and elongate vertebrates.

Introduction

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.

Results

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.

Discussion

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.

Concluding remarks

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.

Acknowledgments

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: tetsuto@ualberta.ca
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