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Comparative reproductive biology of Ariolimax californicus and A. dolichophallus (Gastropoda; Stylommiatophora)

JANET L. LEONARDa; JOHN S. PEARSEa; ALICE BRYANT HARPERb a Joseph M. Long Marine Laboratory, University of California Santa Cruz, Santa Cruz, CA, USA b Bay Leaves Press, Aptos, CA, USA Online publication date: 01 December 2010

To cite this Article LEONARD, JANET L. , PEARSE, JOHN S. and HARPER, ALICE BRYANT(2002) 'Comparative

reproductive biology of Ariolimax californicus and A. dolichophallus (Gastropoda; Stylommiatophora)', Invertebrate Reproduction & Development, 41: 1, 83 — 93 To link to this Article: DOI: 10.1080/07924259.2002.9652738 URL: http://dx.doi.org/10.1080/07924259.2002.9652738

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Invertebrate Reproduction and Development, 41 :1-3 (2002) 83-93 Balaban, PhiladelphiaAZehovot 0168-8170/02/$05.00 0 2002 Balaban

83

Comparative reproductive biology of ArioZimax caZ~ornicusand A. doZichophaZZus (Gastropoda: Stylommatophora)

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JANET L. LEONARD'*, JOHN S. PEARSE' and ALICE BRYANT HARPER' 'JosephM. Long Marine Laboratory, I00 Shaffer Road University of CaliforniaSanta Cruz, Santa Cruz, CA 95060, USA Tel. + I (650) 726-0952; Fax +I (831) 459-3383; email:jlleona@ats. ucsc.edu 2BayLeaves Press, 160 Robideaw Road Aptos, CA 95003, USA Received 20 July 2001; Accepted 19 December 2001

Summary Traits which differ among species in a genus are considered to be the product of relatively rapid evolution. Laboratory studies indicate that copulation differs between two species of the banana slug Ariolimax with A. dolichophallus having a pattern of a single, long duration (typically greater than 1 h) intromission which is simultaneously reciprocal, and A. californicus having mating encounters typically involving a bout of brief (typically 10-20 min) unilateral intromissions. Reciprocal apophallation was observed for both species. Field observations confirm simultaneously reciprocal copulation and apophallation in A. dolichophallus. Mating was more frequent in the spring and summer months. In laboratory studies the two species also differed in life history parameters; mean growth rate was greater in A. californicus than A. dolichophallus, whereas eggs are larger in A. dolichophallus (range = 0.334.5 g; n=59) than in A. californicus (range= 0.08-0.27 g; n=164). Key words: Stylommatophora,sexual behavior, growth, life history, development

Introduction Terrestrial slugs ofthe genus Ariolimm (Stylommatophora: Arionidae) are common and conspicuous members of moist temperate forests on the west coast of North America from San Diego County, California, north to the vicinity of Juneau, Alaska (Harper, 1988). These banana slugs are large (up to 150 g, see below), striking in appearance (often bright yellow or heavily spotted), have a catholic diet (Richter, 1976; Harper, 1988), and a bizarre sexual behavior. At the end of a

copulation one slug may chew off the penis of the partner (Heath, 1916; Mead, 1942, 1943; illustrated in Harper, 1988). This behavior, termed apophallation, was first described by Heath (1916), who noted that approximately 5% of the large A. californicus he collected for use in classroom dissections lacked a penis, suggesting that they belonged to the genus AphaZZarion (Pilsbry and Vanatta, 1896) To resolve the mystery, Heath (1 9 16) erected two outdoor enclosures, filled them with A. californicus and with

'Corresponding author. Proceedings of the Ninth International Congress on Invertebrate Reproduction and Development, Rhodes University, Grahamstown, South Africa, July 15-20, 2001


a4

J.L. Leonard et 01. / IRD 41 (2002) 83-93

the help of his students, eventually observed unilateral copulations and two instances of apophallation. Mead (1 942,1943)determined that Aphallarion was a nomen nudem - previously described specimens being variously attributable to three taxa of Ariolimar; californicus californicw, colwnbianus columbianus, and dolichophallus.Apophallationapparentlydoes not occur in A. californicus brachyphallus or A. columbianus stramineus (Mead, 1943,Pilsbry, 1948). In this paper we compare A. calijornicus californicus, (hereafter, A. californicus) and A. dolichophallus. Although the adaptive significanceof apophallation has been the subject of a great deal of speculation (ex. Heath, 1916;Wright, 1938;Mead, 1942,1943;Pilsbry, 1948; Harper, 1988; Leonard, 1991; Baur, 1998; Birkhead, 2000; Reise and Hutchinson, 2002), the reproductive biology of Ariolimar spp. has attracted relatively little attention (but see Mead, 1942;Wright, 1938;Goitfried and Dorfman, 1970;discussion below) and remains poorly understood. In order to understand the functional significance of apophallation, it will be necessary to improve our understanding of the mating system(s) and reproductive biology of Ariolimar spp. The current study is a first step in this direction.

longer in proportion to the slug than was that of A. californicus, consistent with the descriptions by Mead (1 942,1943)and Pilsbry (1948). Four groups of A. californicus contributed to these observations. Five individuals collected 20-29 January 1999, and seven individuals collected 8 November 2000, were from the vicinity of Purissima Creek, in central San Mateo County, California (“adult californicus”). They were individually housed from their arrival at the laboratory and all were used in behavioral observations. Two more series were comprised of animals hatched from egg clutches laid in the laboratory by individuals of the adult series. A series, consisting of 32 individuals hatched from a clutch of eggs laid by a slug of the “adult californicus” series, appears in the copulation, growth and egglaying data as “lab-hatched californicus ‘‘ (Fig. 2). The copulation data for “lab-hatched californicus” were derived from a subset of these individuals, randomly chosen from the six rearing boxes before 8 June 2000 at less than 20 g weight and individually housed. A third group, “singleton californicus”,appearing only in the growth data (Fig. 2), is a single individual, the only hatchling from a clutch laid by another slug of the “adult californicus” series, reared in isolation from hatching except for behavioral observations.The fourth Materials and Methods group, “lab-reared californicus”, consisted of six individuals collected in Hillsborough, San Mateo County, Field observations 16 May 2001.These individuals weighed less than 2 g Field observations were conducted in Santa Cruz when collected and were individually housed before County, California, primarily in or near the town of reaching 10 g in weight. They were isolated from that Aptos, within the range of A. dolichophallus Mead by time except during behavioral observations. They ABH. Over a 21-year period (1971-1991), pairs of appear in the copulation and growth data. slugs observed mating were photographed with a Individuals of A. dolichophallus Mead were camera which recorded and printed the date, and the collected for laboratory studies from three localities in photographs stored. In some cases notes as to the Santa Cruz County. Eight individuals were collected in duration of the behavior and/or weather conditions Pogonip, six in November 1998 and two more on 18 were made. The slugs were encountered by chance January 1999,the “adult dolichophallus” series. The during the course of daily activities and walks. “lab-reared dolichophallus” consisted of 15 juvenile individuals weighing 2.0-8.9 g, collected in Aptos, 30 April 1999.All individuals of these two series (“adult Laboratory observations dolichophallus” and “lab-reared dolichophallusyy), Animals were individually housed. A third series, “dolichoAll specimens of A. carifornicus Cooper and A. phallus siblings” consisted of 10 individuals hatched dolichophallus Mead were assigned to species on the from a clutch laid by a slug Df the “adult dolichobasis of geography as described in Mead (1943)and phallus” series. These individuals (“grouphoused Pilsbry (1948) and collected well away from a dolichophallus” were group housed from hatching and presumptive contact zone along the San Mateo-Santa appear only in the growth data. In addition, three Cruz border and east into Santa Clara County (Pilsbry, individuals from the vicinity of Rancho del Oso were 1948). Observations of the penis during copulation collected 8 December 2000, group housed at the confirmed that the slugs expected to be A. califrnicus laboratory and appear only in the egg-laying data. Of had a very robust penis whereas the presumptive A. these, one pair was observed copulating, showing the dolichophallushad a much more slender penis that was typical A. dolichophallus form and penis shape.

J.L. Leonard el al. / J R D 41 (2002) 83-93


Maintenance and rearing conditions

Initially the slugs were maintained at ambient temperature in an unheated room in large glass jars lined with moist paper towels and covered with fine wire mesh. Slugs were fed a mixed diet including dog or cat food (kibble), lettuce, and other vegetables. Culture conditions were changed in August 1999 to conform more closely to those used by Deyrup-Olsen (1996). Starting in August 1999, the slugs were housed in plastic boxes, the size determined by the number and size of slugs to be housed. The boxes were cleaned with hot water at least once a week, the floors lined with damp paper towels, and food consistingoforganic lettuce, cat food (kibble) and slices of “yam” (Zpomaea spp), usually red garnet yams, added. Button mushrooms were included less regularly and occasionally other foods were offered. Food was offered ad libitum. The boxes were housed in one of two lightcontrolled rooms, maintained below ambient indoor temperature by the cooling effect of open flow-through seawater tanks (annual range 12-18°C). The annual range of air temperatures in these rooms was 15-20°C. Light cycles in these rooms are controlled by astronomic clocks that follow the annual cycle to be inor 6 months out-of phase with the natural light cycle. Slugs from all series except “lab-hatched dolichophallus” and “lab-reared calfornicus” were assigned so that half were maintained with an in-phase light cycle and half in an out-of-phase light cycle. The “dolichophallus siblings” and the “lab-reared californicus” were maintained in the out-of-phase light cycle. When eggs (or a slug in the process of egg-laying) were found in a box, they were transferred to a clean box lined with damp paper towels (labeled with the date, number of eggs, and identity), and checked at least once a week for signs of development. Near the expected time of hatching, bits of lettuce were added to the box. Eggs that showed no signs of development were retained for at least 12 weeks before being discarded unless they collapsed previously. Eggs were weighed when first found or as soon as possible thereafter. In small clutches (N 41 (2002) 83-93

and head-swinging courtship, (b) intromission, or (c) close contact in the copulation position for >1 min. Copulation was recorded as beginning when a pair of individuals remained in tight contact in the copulation position for >1 min or when at least one penis was observed to connect the pair. The data given here consist of the duration of intromissions only and do not include the length of courtship behavior. Copulations were recorded as unilateral if one and only one penis was visible, reciprocal if two penes were visible at some point in the copulation and uncertain if no penis was visible or if it was unclear whether one or two penes connected the pair. Reciprocal copulations often involved withdrawal of one penis before the other. In that case the duration of the copulation was determined by the last withdrawal. Given the long duration of the observation periods (>lo h in some cases), it was sometimes necessary for the observer to be absent from the room for short periods of time. In that case the videotape record was used to determine the behavior. A mating encounter was defined as an interaction between two or more slugs which included at least one copulation and involved continuous sexual behavior.

Results

Sexual behavior Over the 2 1 years, A. dolichophallus were observed mating in the field a total of 15 times. The majority of matings were observed in the summer months (JuneSeptember) with others in the spring (February-April)

(Fig. 1). Notes were made for three of the matings. On 14 September 1977, an overcast day, a pair were observed to mate for more than 5 h. On 2 June 1987, a pair were found mating at 10 am and reported as trying to separate at 9:30 pm. On 8 August 1987, in foggy conditions, a pair was found at a pre-mating stage at 10:30 am and in the process of unilateral apophallation at 3 pm. In six of these observations, the copulations were clearly simultaneously reciprocal. One hundred and twenty-one copulations were observed and recorded in their entirety in the laboratory, of which four involved apophallation: three for A . dolichophallus and one in A. californicus (Table 1). There was one heterospecific mating, where an A . californicus (“adult californicus” series) got in between two courting A . dolichophallus of the “adult dolcihophallus” series. The heterospecific copulation consisted of a simultaneously reciprocal intromission lasting 19 min. It was followed within 10 min by a simultaneously reciprocal intromission between the A. dolichophallus involved in the heterospecific mating and the conspecific that it had been previously courting, which lasted 113 min. The heterospecific copulation was not included in the quantitative data summaries (Tables 1-3). Both individuals involved in the heterospecific mating subsequently laid eggs and the offspring will be tested for paternity at a future date. The courtship behavior observed for both species in this study involved (1) exploratory contact, progressing to (2) an antiparallel orientation, and (3) reciprocal head-swinging and biting by the pair, accompanied by

No apophallation observed 0With single apophallation With reciprocal apophallation

m

>

I‘

rJ E’

z3

0

Fig. 1. Copulations, single apophallations and reciprocal apophallations in Ariolimax dolcihophallus observed in the field, near Aptos, California, for each month of the year, over a 21-year period.

J.L. Leonard et al. / IRD 41 (2002) 83-93

87

Table 1. Copulation summary dolichophallus

No. of individuals Mean duration, min Standard deviation No.of copulations No. of pairs No.of encounters No.of apophallations

Californicus

Adults

Lab-reared

Adults

Lab-reared

Lab-hatched

6

6 209 174 13 8 13 1 reciprocal 1 unilateral

6 64 89 14 4 12 1 reciprocal

3 24 16 12 3 9 0

9 14 12 73 17 31 0

145 85 7 6

7 1 unilateral


Table 2.Types of intromission for A . californicus and A . dolichophallus Species

Simultaneously reciprocal intromission

Unilateral intromission wlo known aphallate involved

Unilateral wl known aphallate

Uncertain

A . dolichophallus

11

3

5

1

(n=20) A. californicus (n=99)

3 (asymmetrical, see text)

35

0

61

Table 3. Comparison of bout lengths for A . californicus and A . dolichophallus

_ _

__

Adult californicus

Lab-reared californicus

Lab-hatched californicus

Adult dolichophallus

Lab-reared dolichophallus

Single copulation Two copulations

8 2

12 9

7 0

13 0

Three copulations Four copulations Five or more copulations

2

3 4 (1 with change of partners) 1 (changed partners) 0 0

0 0 0

0 0 0

0 0

gradual swelling of the genital regions, and (4) the genital regions of the pair came into contact and then separated a few times before the pair rested motionless with the swollen genital regions in alignment. Copulation was considered to occur when the pair remained motionless in this copulatory posture for 1 min or more, or at least one penis was seen connecting the pair. Two types of copulation were observed in this study: simultaneously reciprocal, in which the pair were connected by two penes for at least part of the copulation duration, and unilateral, in which only one penis connected the pair. In many cases, the genital areas were so closely apposed that it was impossible to determine conclusively whether the copulation was unilateral or simultaneously reciprocal. Out of 20 copulations between pairs of A. dolichophallus, 1 1 were simultaneously reciprocal,

2 0

7

eight were unilateral, and 1 was uncertain (Table 2). Of the eight unilateral copulations, known aphallate individuals (individualswhose apophallation had been observed earlier) were involved in 5. In the brief copulations typical of A, californicus, particularly of the “lab-hatched californicus” (Table l), the penis was often not visible and for 61 of the 99 copulations the type of intromission was unclear. Of the remainder, 35 were unilateral, none involving a known aphallate animal, and 3 were simultaneously reciprocal (Table 2). Where reciprocal intromission was observed between pairs of A. californicus, it was asymmetrical, with part of the thick end of one penis and part of the thin end of the other visible between the partners. In A. dolichophallus the visible portions of the two penes were comparable in thickness for reciprocal copulations.

88

J L. Leonard et al. / IRD 41 (2002) 83-93

Copulation duration was measured from the beginning of copulatory posture to the time when no penis was connecting the pair. Copulation durations recorded in this study varied over a wide range from the minimum criterion of 1 min to the record of 710 rnin (the reciprocal apophallation described below). The means of the different groups (Table l), ranked from lowest to highest are: (1) “lab-hatched californicus,5 = 14 min; (2) “lab-reared califomicus”, = 24 min, (3) “ adult calfornicus”,x = 64 min; (4) “adult dolichophallus”,? =145, and (5) “lab-reared dolichophallus”,X = 209 min. Copulations terminated either (1) with smooth withdrawal, (2) after a prolonged period of crawling and turning with great distension and stretching of the penis before it finally is free of the female aperture, or (3) with apophallation. Four instances of apophallation were observed in this study. In apophallation, one or both slugs turned toward the pehis area during copulation and began chewing on the partner’s penis. In order for chewing to occur there had to be a sufficient length of penis exposed between the pair to allow a slug to turn its head far enough to get its radula over the penis. Once effective chewing began, one could determine whose penis was being attacked by the violent writhing movements of the owner. Autoapophallation, as reported by Mead (1942) for A. dolichophallus, was not observed in this study. In each case the penis was severed by the partner. Apophallation was a slow process and normally the gradual erosion of the penis is visible before the penis is severed. Once apophallation is complete, the severed stub of the penis is usually eaten by the animal who severed it. Apophallation is a lengthy process and copulations involving apophallation are usually of long duration (Table 1). The one apophallation observed for californicuswas reciprocal, with the copulation lasting 268 rnin to the first amputation, and the second penis severed 65 rnin later. Of the three apophallations observed in dolichophallus. two occurred in the “lab-


x

reared dolichophallus” with one being reciprocal, lasting 329 rnin until the first amputation, with the second penis severed 381 rnin later, for a total copulation duration of 7 10 min. The copulation which ended in unilateral apophallation for this group lasted 380 min; and the fourth apophallation, in the “adult dolichophallus” group, was also unilateral and the copulation lasted 255 min. All of the mating encounters observed between A. dolichophallus in this study consisted of a single copulationwith the pair separatingthereafter (Table 2). The one time an A. dolichophallus was involved in more than one copulation during an observation period, the first copulation was heterospecific (see above) followed by a .second homospecific copulations. In A. calfornicus, out of 52 encounters resulting in copulation, 23 consisted of a single copulation and 18 involved multiple copulations. One bout observed in “lab-hatched calfornicus” consisted of a series of eight copulationsbetween the same pair of individuals; there were seven bouts of more than five copulations (Table 3). In a few cases the members of the pair were observed to alternate sexual roles, and, in two cases of bouts of three copulations, the pair copulated with A male and B female, then performed a simultaneously reciprocal copulation and then copulated again with A female and B male. In the majority of cases the roles were not clear. In the “lab-reared californicus”,there were two cases in which one of the members of a pair mated within 30 min with a third individual, creating encounters with a change of partners. In the few encounters in this study in which both individuals were known to be virgins, the one pair of A. dolichophallus copulated for 53 1 min, with reciprocal intromission; whereas two of the “lab-hatched cailfornicus” pairs showed single unilateral copulations (30 and 28 min). A virgin pair of “lab-reared californicus” copulated unilaterally for 12>min, and then the first female mated as a male with a third individual (also a virgin) for 28 min, with the roles unclear.

Table 4. Egg clutches laid Group

No. of individuals laying eggs

No. of c1utches

No. of clutches whatchlings

Hatching success, %

Copulated in lab before egg-laying

Adult dolichophallus Lab-reared dolichophallus Group-housed dolichophallus Adult colifornicus

2 0 1

-

2

1 -

-

62

2 6

1

6

1 4

29

4

Probably 3

Lab-reared calvornicus Lab-hatched californicus Total

0 8+

-

-

-

14

8

23

7 13

14

15

14-35 (change)


J.L. Leonard el al. / IRD 41 (2002) 83-93

1 3 6

89

7 0 11 1 3 1 5 1 7 1 9 2 1 2 3 2 5 ~ ~ 3 1 3 3 3 6 3 7 3 9 4 1 4 3 4 6 4 7 4 9 6 1 ~ 3 ~ a t 0 1 e 3 ~ ~ 7 ~ 7 1

Age h weeks Fig. 2. Changes in weight of five groups of juvenile slugs raised in the laboratory. The age at capture of the “lab-reared dolichophallus” and “lab-reared californicus” groups was estimated by their average weight as compared to the average weight of the “dolichophallus siblings”, “singleton californicus”,and “lab-hatched californicus”. For each group n is given as the number of individuals at the start of the study. The standard deviation of the mean is shown by error bars until the number declines to below eight individuals. The mean is plotted as long as two individuals remain. The “singleton californicus” data shows the weight of one individual throughout the observation period. The “lab-hatched californicus ” and the “dolichophallussiblings” both suffered high mortality in February and March 200 1, truncating the growth curves for those groups.

Growth pailerns The growth rates of the two groups of A. dolichophallus and the “singleton californicus” were very similar (Fig. 2). By contrast growth of the “lab-hatched californicus” and “lab-reared californicus” was more rapid (Fig. 2). The larger standard deviations reflect a broader range of growth rates in the “lab-hatched californicus”.Some individuals in this group showed a pattern of weight increase comparable to that of the “singleton calijornicus”,whereas others reached over 75 g at a time when the smallest of the group were under 25 g. The pattern of mean weight increase of the “lab-reared cailfornicus” was similar to that of the “lab-hatched californicus”. Egg laying Animals produced a total of 23 egg clutches, three by A. dolichophallus and 20 by A. californicus slugs. The clutch size for A. californicus varied from 1 to 62 eggs; the three A. dolichophallus clutches contained 17-22 eggs (Fig. 3). Thirteen of the clutches were laid

Fig. 3. Clutch size as a function of mean egg weight for pooled data from Ariolimax californicus and A. dolichophallus. The standard deviation of the mean weight is shown as error bars.

by slugs of the “lab-hatched calrfornicus ” series. Weights were obtained from two A. dolichophallus clutches, whose parents were derived from two distinct populations and from 17 A. californicus clutches all derived from a single population. The eggs

90

J. L. Leonard et al. / IRD 41 (2002) 83-93

Table 5. Other life history parameters -_

Trait

-

-~

~

~

_

Min. time between clutches Max. number of clutches Max. time from last copulation to egg laying, months Max. time from last copulation to egg laying with viable hatchlings, months Max. recorded weight, g Max. age ~.

-

_

A. californicus

__..

.___

0.33-0.50 17-22 (n=3) 51-55 days (n=2) Colorless Approx. 13 months from hatching Mean: 37.6 g (n=6); range: 21 S 4 9 . 4 1 g Approx. 15 months afier hatching, 2 months after 1st copulation, 2nd encounter wlcopulation for pair, 3rd copulation for one individual, 2nd for the other Unknown ??; all collected as adults; Ranch del Oso, 38.2 or 26.2 g; #6, Pogonip slug approx. 60.30 g; #3, Pogonip slug 41.6 g Unknown 1

Age at first egg-laying Weight at first egg-laying Downloaded By: [Stanford University] At: 23:13 21 June 2011

_

A. dolichophallus ~

Egg size, g Range of clutch size Time to hatching Color of hatchlings Min. age at first copulation Weight at first copulation Earliest observed apophallation

~.

~

~.

___

~~~

0.084.27 1-62 (n=21) 46-81 days (n=lO) Colorless 8 months from hatching Mean: 5 1.44 g (n=9); range: 43.12-6 1.19 Collected as adult

50 weeks from clutch laid Individually housed, lab-hatched californicus, = 60.92, max= 74.33, min =52.88. n=6

x

5

6 days 4 for .isolated individuals; one box of four produced 5 egg masses 9

2

7.5

60.3 #6, collected as adult; lived 19 months in lab

149 GG collected as adult; lived 25 months in lab

__

__.

__

.-

of A. dolichophallus ranged from 0.33 to 0.50 g in weight (n=37). For A. californicus egg weights ranged from 0.08 to 0.27 g (n=397). The time to hatching of the two A. dolichophallus clutches (51-55 days) fell midway in the range of the A. californicus clutches (46-81 days) (Table 5). Hatchlings of both species were colorless as previously reported (Harper, 1988). The youngest animal to lay eggs in this study was 50 weeks old, measured from the time its birth clutch was laid. None of the lab-reared or hatched A. dolichophallus laid eggs during this study. Table 5 gives a comparison of the two species for a variety of reproductive and life history parameters.

__

__

__

-__

~.

_-

common period for copulation” in the field was August and September. Moreover, I. Deyrup-Olsen (pers. comm.) found that in a laboratory population of A . columbianus collected in Washington State, mating occurred in the late summer with egg-laying in the autumn and early winter. The field observations reported here are also the first observation of reciprocal apophallation in Ariolimax, and demonstrate that copulation in A. dolichophallus typically involves simultaneously reciprocal intromission, consistent with the laboratory observations, rather than unilateral as reported by Mead (1 942). Mead (1942) observed two copulations in A. dolichophallus, both being unilateral, but each copulation involved one aphallate individual.

Discussion

Field observations for A. dolichoDhallus The mating observations reported here represent the first field data on the mating- season for any Ariolimax species. The spring and summer mating season found for A . dolichophallus is consistent with Wright’s (1938, p. 9) report for A . californicus, that the “more

~

.

Laboratory observations Egg size and growth rate All eggs of A. dolichophallus were heavier than those of A. californicus and the growth rates of the A . dolichophallus were both slower on average and less


J.L.Leonard et al./ IRD 41 (2002) 83-93 variable than observed in even a single clutch of A. californicus (“lab-hatched californicus”).Considerable within-clutch variation in growth rate is not unusual in pulmonates (South, 1992; Heller, 2001) and may not reflect genetic variation (Shibata and Rollo, 1988). In this study, larger egg size was associated with slower growth between species, whereas Shibata and Rollo (1988) reported that larger egg size was associated with slower growth rates within a clutch. There did not appear to be any relationship between the number of eggs in the clutch and mean egg weight for A. californicus. Although theory has assumed that there will be a trade between clutch size and egg size (Steams, 1992; Heller, 200 l), empirical data do not in general support such a trade-off, suggesting that energy for egg production is not an isolated pool (review in Stearns, 1992). Heller (2001) reported that across species maximum animal length (or shell diameter) was correlated with egg length for stylommatophorans. Since we considered animal length to be a less reproducible measure than weight, we did not measure length in this study. Both species commonly reach lengths of 20 cm or more. Egg length was not measured. The hatching time was similar for the two species in this study, perhaps because they were reared under very similar laboratory conditions. Previous studies have suggested that temperature is of paramount importance in determining development time in stylommatophorans. Life span and sexual maturity In a previous laboratory study, Gottfried and Dorfmann (1970) followed gonadal development in lab-reared A . californicus from a population in Santa Clara County, over a period of 2 years and described a young male stage of the gonad as appearing at an estimated age of 6 months and 20 g in body weight. In our study the first copulation for lab-hatched A . californicus occurred at a minimum age of 8 months and a mean body weight of 51 g. Furthermore, these authors described the gonad as reaching an intersex (both sperm and eggs present) stage at an age of 24-30 months and weight of 45 g. In our observations, an A. californicus laid eggs at an age of 50 weeks from the time its clutch was laid and although hatching success was low for eggs laid by “lab-hatched californicus”, some eggs did develop normally, indicating that the mother was reproductively mature. The differences in maturation times may reflect a difference in rearing conditions (Cunningham and Gottfried, 1967) or population differences. Self-fertilization is not the rule in stylommatophorans (Tompa, 1984; Runham

91

and Hunter, 1970; South, 1992; Heller, 2001) but cannot be ruled out for Ariolimar. Mead (1 942) raised Ariolimax in isolation for more than 18 months but did not observe egg production so that there is at present no evidence of self-fertilization or apomixis in Ariolimar. Paternity analysis will be required to determine whether the hatchlings from these clutches were the product of cross- or self-fertilization, as well as whether heterospecific copulation results in viable offspring. Sexual behavior The most significant conclusion of this study is that A. californicus and A. dolichophallus differ in mating system. In A. californicus, sexual encounters often involved a bout (series) of copulations between a pair of individuals (Table 3), usually with unilateral intromission (Table 2). This confirms previous reports of unilateral intromission in A . c. californicus. (Heath, 1916; Wright, 1938) but is the first report of bouts of copulation in A . c. californicus. Mead (1942) describes one observation of copulation in A. c. brachyphallus in the field involving of a bout of unilateral copulations with alternating sexual roles. Bouts of mating with alternation of sexual roles is a familiar type of mating system in simultaneous hermaphrodites (Fischer, 1980; Leonard and Lukowiak, 1984; Sella, 1985; Ramos et al., 1995; reviews in Leonard, 1991, 1993, 1999; Michiels, 1998; Petersen, 1990). Bouts of mating without role alternation are not well known and would be a novel type of mating system. The application of sexual selection theory to hermaphrodites (Axelrod and Hamilton, 1981; Charnov, 1979; Leonard, 1990) predicts that mating systems in simultaneous hermaphrodites will involve reciprocity between the pair to resolve sexual conflict over the preferred sexual role. Theory therefore predicts that alternation of sexual roles during bouts of copulation will be the rule rather than the exception in A. californicus, but further study is required to test this prediction. In A . dolichophallus, sexual encounters in this laboratory study typically involved a single copulation between a pair of individuals, with simultaneously reciprocal intromission. The portions of the penes visible between the pair are similar in thickness in this species. Reciprocal intromission was also observed in A. dolichophallus in the field. The majority of observed unilateral copulations in the laboratory in this species involved copulations in which an intact individual acted as male to another individual known to be aphallate. Further our data demonstrate that reciprocal as well as unilateral apophallation occurs in both


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species. In addition, two laboratory observations of copulation in A. columbianus involved simultaneously reciprocal copulation with equal size penes visible (Leonard and Pearse, unpublished). The sexual biology of Ariolimax is of particular interest for two reasons. The first is the insight it may offer into speciation in this group and by analogy in stylommatophorans in general . The pattern found in Ariolzmax of taxa being distinguishable at the species level primarily by characters of the genitalia is common in many taxonomic groups, among them the stylommatophoran gastropods, and Eberhard (1985) has hypothesized that this rapid evolution of genitalic traits is evidence for sexual selection. The role of sexual selection in the speciation and evolution of the Stylommatophorahas become of increasingimportance to taxonomists in recent years (e.g., Emberton, 1994; Roth, 1996; Reise, 1996; Jordaens et al., 1998). A second and perhaps even more compelling reason to learn more about the sexual biology of Ariolimax is the opportunity it may offer to test current theories of sexual selection and sex allocation. The extension of sexual selection theory to hermaphrodites is attributable to an initial suggestion by Bateman (1948), with much fuller treatment by Ghiselin (1974) and Charnov (1979; 1983). Sexual selection in simultaneous hermaphroditeshas become an exciting field in recent years both because of its potential to test theory developed in dioecious organisms and its potential for understanding the functional significance of some of the bizarre sexual habits of invertebrate hermaphrodites (reviews in Ghiselin, 1974; Leonard, 1991, 1993, 1999; Baur, 1998; Michiels, 1998; Premoli and Sella, 1995). This study demonstrates that apophallation is a component of two distinct mating system in Ariolimax spp. The discovery that A. californicus and A. dolichophallus exhibit different mating systems which include apophallation indicate that at least three mating systems have evolved in the five taxa of this genus, since apophallation is known to be absent in two of the taxa. This genus then offers great potential for comparative studies in the evolution and mating behavior of internally fertilizing hermaphrodites comparable to those in serranid fishes (reviews in Petersen, 1990; Leonard, 1993). Further work describing the circumstances under which apophallation occurs in each species, along with descriptions of the sexual behavior and mating system of those species of Ariolimax which do not perform apophallation, will allow us to both understand the functional significance of this behavior and use it to test current theories of sex allocation and sexual selection.

Acknowledgements We wish to thank Ingrith Deyrup-Olsen for her advice, help, and encouragement with all aspects of slug culture; Barry Roth for advice and encouragement with various aspects of ariolimacine biology; Dave Casper for supplying juvenile dolichophallus and helpful discussions; Vicki Pearse and Louise Pearse for help with various aspects of the study; Ken Vicknair for collecting juvenile californicus; Daniel Harper for support with field observations and photography, and Betsy Steele and the staff of the Long Marine Laboratory for their support with space and facilities. This research was supported in part by a UCSC Faculty Research Grant to JSP. References Axelrod, R., and W.D. Hamilton, The evolution of cooperation. Science, 21 1(1981)1390-1396. Bateman, A.J., Intra-sexual selection in Drosophila. Heredity, 2 (1948) 349-368. Baur, B., Sperm competition in molluscs. In: Sperm Competition and Sexual Selection, T.R. Birkhead and A.P. Msller (eds.), Academic Press, New York, 1998, pp. 255-305. Birkhead, T., Promiscuity. Harvard University Press, Cambridge, MA, 2000. Charnov, E.L,. Simultaneous hermaphroditism and sexual selection. PNAS, 76 (1979) 2480-2484. Charnov, E.L, The Theory of Sex Allocation. Princeton University Press, Princeton, N.J., 1983. Cunningham, J.J. and Gottfried, H., The laboratory care of giant land slugs. Lab. Animal Care, 17 (1967) 382-385. Deyrup-Olsen, I., Product release by mucous granules of land slugs: 11. Species diversity in triggering of mucous granule rupture. J. exp. Zool., 276 (1996) 330-334. Eberhard, W.G., Sexual Selection and Animal Genitalia. Harvard University Press, Cambridge, MA, 1985. Emberton, K.C., Poylgyrid land snail phylogeny: external sperm exchange, early North American biogeography, iterative shell evolution. Biol. J. Linn. Soc., 52 (1994) 24 1-27 1. Fischer. E.A., The relationship between mating system and simultaneous hermaphroditism in the coral reef fish, Hypoplectrus nigricans (Serranidae). Anim. Behav., 28 (1980) 620-633. Ghiselin, M.T., The Economy ofNature and the Evolution of Sex. University of California Press, Berkeley, CA, 1974. Gottfried, H. and Dorfman, R.I.,Steroids of invertebrates IV. On the optic tentacle-gonadal axis in the control of the male-phase ovotestis in the slug (Ariolimaxcalgornicus). Gen. Comp. Physiol., 15 (1970) 101-119. Harper, A.B., The Banana Slug. Bay Leaves Press, Aptos, CA, 1988. Heath, H., The conjugation of Ariolimax californicus. Nautilus, 30 (1916) 22-24.

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Steams, S.C., The Evolution of Life Histories. Oxford University Press, Oxford, 1992. Tompa, A.S., Land snails (Stylommatophora). In: The Mollusca, Vol. 7, Reproduction, A S . Tompa, N.H. Verdonk and J.A.M. van den Biggelaar (eds.), Academic Press, Orlando, 1984, pp. 47-140. Wright, C.C., A Study of Reproduction and Sex Determination in Ariolimax californicus Cooper. PhD Dissertation, Stanford University, 1938.