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Biology Faculty Publications and Presentations

1-2016 SEXUAL VS. ASEXUAL IN A STICK INSECT (MEGAPHASMA DENTRICUS) Tara Maginnis University of Portland

Christopher R. Redmond

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Citation: Pilot Scholars Version (Modified MLA Style) Maginnis, Tara and Redmond, Christopher R., "SEXUAL VS. IN A STICK INSECT (MEGAPHASMA DENTRICUS)" (2016). Biology Faculty Publications and Presentations. 42. http://pilotscholars.up.edu/bio_facpubs/42

This Journal Article is brought to you for free and open access by the Biology at Pilot Scholars. It has been accepted for inclusion in Biology Faculty Publications and Presentations by an authorized administrator of Pilot Scholars. For more information, please contact [email protected]. TEXAS .1. OF SCI. 65(1 ):3-l3 FEBRUARY, 2013 (PUBLISHED JAN 2016) SEXUAL VS. ASEXUAL REPRODUCTION IN A STICK INSECT (.MEGAPHASMA DENTRICUS)

Tara Maginnis1 and Christopher R. Redmond2 ‘Department of Biology, University of Portland 5000 N. Willamette Blvd, Portland, OR 97203 2St. Edward's University, Department o f Natural Sciences 3001 S. Congress Ave., Austin, TX 78704

Abstract. -The paradox of is one of biology’s great evolutionary questions, particularly in those that are fully capable of sexual and asexual reproduction. To quantify how varies between these two modes of reproduction, we explored lifetime fecundity in Megaphasma dentricus, the giant walking stick ofNorth America. For the first 20 days of egg laying, there were no fecundity differences between mated and unmated with respect to egg number or egg weight; all females laid a total of ~50 eggs and each egg weighed about 0.02g. For days 21-50 (the last 30 days of egg laying), unmated females laid significantly fewer (but not lighter) eggs than sexually reproducing females. Overall, lifetime fecundity in unmated females was about 5-10% less than mated females. Myriad factors remain unexplored in this species, including the of sexually and asexually produced eggs, the effects of parasites or other considerations of co- (e.g., the ), and the accumulation of deleterious (e.g., Muller’s Ratchet).

Sex is one of evolution’s greatest innovations. However, it is also one of the most paradoxical, given the ultimate costs of (Maynard Smith 1971; Williams 1975; Lloyd 1980; Michod & Levin 1988; Crow 1994; Misevic et al. 2009; Roze 2012). For example, sexual reproduction can destroy favorable allelic combinations during , and compared to asexual reproduction, sexually reproducing organisms only contribute half of their genetic material to offspring. In addition, unmated females who reproduce asexually may have a higher fitness because they can produce twice as many offspring compared to sexuals, and thus quickly replace sexually reproducing individuals (e.g., Maynard Smith’s the “cost of males” hypothesis, 1978).

Despite potential costs, most reproduce sexually. One of the major evolutionary explanations is ; given that an obligate, asexually reproducing population is destined to be a tip on the tree of , the reshuffling of genetic material through sex 4 THE TEXAS JOURNAL OF SCIENCE-VOL. 65, NO. 1, 2013 helps maintain the raw material for selective processes and potential (Muller 1964; Felsenstein 1974; Williams 1975; Bell 1982; Otto & Lenormand 2002; Melian et ah, 2012; Song et al. 2012). Two other fundamental hypotheses for the evolution and persistence of sexual reproduction include the “Red Queen” and “Muller’s Ratchet”. Counter to the short-term fitness benefits of asexual reproduction, clonal populations may be unable to escape any long-term co- that has specialized on their single genotype (Van Valen 1973; Stenseth 1979; Hamilton et al. 1990; Ridley 1995). For example, in the Red Queen Hypothesis a population of genetic clones would be unable to escape a parasite, leaving the host organism unable to evolve and adapt. Similarly, without the influx of new, recombinant regions of DNA in each generation, deleterious mutations cannot be fragmented and are likely to become fixed in the population. In the long-term this irreversible (and unidirectional, like a ratchet) accumulation of maladaptive genetic combinations can drive a clonal population to (Muller 1932; 1964; Felsenstein 1974).

Although sexual reproduction is the dominant mode of reproduction in the clades, some taxa are capable of both sexual and asexual reproduction. Walking sticks (Order ) are fairly unique in that for many species, females can readily reproduce both sexually and asexually despite a potentially healthy proportion of males (Bergerard 1958; Giorgi 1992; Brock 1999; Otte & Brock 2005). Megaphasma dentricus (Stal 1875), the giant walking stick of North America, is one such species. It in wooded areas of Texas and Oklahoma as well as select regions of Mexico (Caudell 1903; Arnett 2000; Otte & Brock 2005). Although field measures of fecundity are ideal (as there are likely myriad contributing factors), M. dentricus does not lay eggs in any type of nest but simply drops them to the ground (prohibiting reasonable data collection in the field). Thus, through a laboratory investigation we quantified lifetime fecundity in both mated and unmated M. dentricus females. Specifically, we aimed to quantify any fecundity differences in these two modes of reproduction (measured via egg MAG1NNIS & REDMOND 5 number and egg weight) to determine if and how asexual reproduction may be affecting long-term fitness in this species.

M ethods Thirty adult females and 30 males were collected in Blanco Co., TX. Eggs from their sexual copulations were kept at room temperature in local soil, misted with water bimonthly, and hatched two years later (e.g., they have a two year incubation, personal observation). From March-April nymphs were reared together in large cages (~1 m3) to minimize leg events due to molting complications, as investing into leg regrowth can negatively impact fecundity (see Maginnis 2006; Bely & Nyberg 2010 for review). Upon their penultimate instar, when the are easily distinguished (late April), the sexes were separated to ensure virginity, and individuals with regenerated legs were removed from the experiment. After maturing (early-mid May), animals were randomly placed into one of two treatment groups: unmated (females never exposed to males) or sexual (a monogamous male and pair). Although polygyny is common in M. dentricus (Maginnis et al. 2008), we did not have enough individuals to create a third treatment group with one female and two males.

Animals were kept in 12 L plastic containers covered with netting, fed Celtis laevigata {ad libitum), misted with water daily, and kept in a rooftop greenhouse exposed to natural light and day cycles. All eggs were collected, counted, and collectively weighed every day for 50 d between the hours of 4-7 p.m. 50 d was chosen as the end point of the experiment for two reasons. First, most of the females died at day 53 or later (see Results). Second, years of collecting trips to Blanco County suggest a 30-50 d adult life span; adults appear in mid-May and can be found in abundance throughout June, but no individual has been collected past July (Maginnis, personal observation). Thus, both the lab and the field support 50 d as a close approximation of lifetime fecundity. 6 THE TEXAS JOURNAL OF SCIENCE-VOL. 65. NO. 1, 2013

To look for fecundity differences between mated and unmated females, we first determined whether or not fecundity was dependent on body size (measured as both body mass and body length). Periodic measurements of female body mass during the first month of their reproductive period revealed that weight is extremely variable (as at any given sampling point females could be unpredictably carrying one to ten eggs, each weighing -0.02 g) and hence a poor measure of body size. Thus, for the final analyses, treatment groups were compared using ANCOVAs with body length as the covariate (measured from the most anterior part of the head to the most posterior portion of the abdomen, not including cerci [see Caudel 1903 for an anatomical reference]). A post-hoc analysis revealed that mated and unmated females did not significantly differ in body length (t-testunequal variance, P =0.63; X± S.D. unmated = 111 .8±10.0 mm, mated = 113.4 ± 14.6 mm).

We also determined whether there was a pattern to the number or weight of eggs laid per day over the 50 d period, and whether potential fecundity patterns differed between treatment groups. Two correlations were done for each female (egg number / over time and egg weight /over time), and the resulting r-values were compared across treatment groups with an unpaired t-test assuming unequal variance. All individual r-values were between -0.41 and 0.03, and -75% of the values were between -0.10 and 0.03 (data not shown). In addition, there were no differences between the treatment groups for either egg number (W q u a i variance , P 0.23) or egg weight (W q u a i variance — , p ~ 0.27). Since there was no robust pattern to the number or weight of eggs laid over time, averages were not used for our final comparisons among treatment groups. Instead, the sum of egg number and the sum of egg weight were compared on days 10, 20, 30, 40, and 50. This was done for both individual sets of 10 d intervals (e.g., days 1-10, 11-20, 21-30, 31-40, and 41-50) as well as cumulative sets of 10 d intervals (e.g., days 1-10, 1-20, 1-30, 1-40, and 1-50). MAGINNIS & REDMOND 7

Results Out of the 52 of 73 nymphs that survived to maturity, 35 were used in this experiment; Mmmated = 11 females and AWed = 24 (12 females and 12 males). All other individuals were either used for breeding stock or were removed from the experiment due to regeneration (as leg regeneration impacts fecundity). Most of the females died after day 53, but one unmated female died on day 24, and one mated female died on day 22. These two females were analyzed for number and weight of eggs from days 1-10, 11-20, and 1-20, but were excluded for all other analyses. One male died day 44, so his female partner was excluded for day 1-50 and 41-50 analyses. Any individuals still alive by day 63 (N = 9 out of 35) were euthanized by freezing and included in all analyses.

There were considerable differences between the two treatment groups with respect to the early and latter periods of reproduction. During the first 20 days of egg laying, there were no fecundity differences between mated and unmated females (all p > 0.263, see Tables 1 and 2). However, after day 20, sexually reproducing females laid more eggs than unmated females (all p < 0.05 except for days 41-50 [p = 0.061], see Tables 1 and 2). There were generally no differences in egg weight between the early or latter reproduction periods, or between the two treatment groups (all p > 0.05 except for days 21-30, see Tables 1 and 2).

Discussion The results of this study suggest that unmated (and thus asexually reproducing) M. dentricus lay fewer eggs than sexually reproducing females, but only during the latter period of reproduction. That unmated females lay fewer eggs than mated females is consistent with some previous studies on geckos (Kearney & Shine 2005), salamanders (Uzzel 1964), millipedes (Enghoff 1976), and (Weinzierl et al. 1999); see Soumalainen (1962) and Lynch (1984) for early reviews. However, the lack of a fecundity difference between the two treatment groups during the first 20 days 8 THE TEXAS JOURNAL OF SCIENCE-VOL. 65. NO. 1, 2013

Table 1. Cumulative fecundity data for mated and unmated M dentricus females over the course of 10, 20, 30, 40, and 50 d. (X ± S.D. and ANCOVA statistics reported).______unmatcd mated unmated mated total # of eggs total # eggs total egg weight (g) total egg weight (g)

23.6 (±9.5) 26.3 (±4.4) 0.434 (±.18) 0.473 (±.06) Days 1-10 N= 11 N= 12 N= 11 N= 12 F ( l, 50) = 0.927p = 0.347 F (l, 0.014) = 0.868 p = 0.363

51.8 (+17.7) 55.2 (±7.2) 0.932 (±0.35) 0.997 (±0.13) Days 1-20 N= 11 N= 12 N = 11 N— 12 F (1, 126) = 0.893 p = 0.356 F( 1, .057) = 1.325 p = 0.263

74.6 (±20.6) 83.7 (±8.7) 1.373 (±0.50) 1.529 (±0.22) Days 1-30 N= 10 N= 11 N= 10 N= 11 F ( l, 693) = 4.539p = 0.047* F (1,0.257) = 3.235 p = 0.089

94.1 (±26.6) 109.3 (±14.9) 1.764 (±0.67) 2.012 (±0.37) Days 1-40 N= 10 N= 11 N= 10 N= 11 F ( l, 1765) = 6,019p = 0.025* F (1,0.589) = 3.538 p = 0.076

113.2 (±34.5) 134.4 (±19.9) 2.093 (±0.85) 2.504 (±0.47) Days 1-50 N= 10 N= 10 N= 10 N= 10 F ( l, 2913) = 5.225 p = 0.035* F (l, 1.192) = 3.835 p = 0.067

Table 2: Fecundity data for individual 10 d periods in mated and unmated M. dentricus females (X ± S.D. and ANCOVA statistics reported, see Table 1 for Day 1-10 results). Unmated total # mated unmated mated o f eggs total # eggs total egg weight (g) total egg weight (g) 28.1 (±13.8) 28.8 (±5.0) 0.503 (±0.27) 0.523 (±0.11) Days 11-20 7V= 11 N= 12 A = 11 N= 12 F( 1, 17) = 0.212p = 0.65 F ( l, .012) = 0.467p = 0.502

20.4 (±7.1) 27.5 (±5.1) .403 (±0.18) 0.520 (±0.13) Days 21-30 N= 10 N= 11 N= 10 N= 11 F ( l, 318)= 10.19/7 = 0.005** F ( l, .10) = 6.144p = 0.023*

19.5 (±7.6) 26.0 (±7.7) 0.396 (±0.18) 0.482 (±0.17) Days 31-40 N= 10 N= 11 N= 10 N= 11 F (1,246) = 4.984 p = 0.039* F (1, .062) = 2.705 p = 0.117

19.1 (±8.7) 25.1 (±6.0) 0.354 (±0.19) 0.470 (±0.19) Days 41-50 N= 10 N= 10 N= 10 N= 10 F (l. 209) = 4.015 p = 0.061 F ( l, 0.082) == 3.772 p = 0.069 MAG1NNIS & REDMOND 9 of their reproductive period is consistent with a related species, the Vietnamese walking stick (Clitumnus extradentatus, now referred to as Baculum extradentatum), where unmated females laid just as many eggs as sexual females for the entire test period of 90 days (Bergerard 1958). With few studies quantifying the variation between sexual versus asexual reproduction in phasmids (Lamb & Willey 1979), if and how M. dentricus and B. extradentatum are representative of this diverse groups remains a mystery.

Interestingly, there were generally no differences in egg weight; regardless of how many eggs each female laid, those eggs were all approximately the same size. For example, if a mated female lays 135 eggs in her lifetime and they collectively weigh a total of 2.5 g (see Days 1-50, Table 1), this means that on average, her eggs weighed -0.02 g each (2.5 / 135 = 0.0185). Similarly, if an unmated female lays 113 eggs, collectively weighing 2.1 g (see Days 1-50, Table 1), her eggs also averaged -0.02 g each (2.1 / 113 = 0.0185). The one exception to the statistical insignificance between the two treatment groups was during days 21-30 where eggs from unmated females were significantly lighter than eggs from mated females. We have no explanation for these results and/or hypotheses on why there would be a significant decrease in egg weight for those 10 days. Regardless, it is likely that stored lipids, proteins, etc. greatly contribute to egg success over their two-year incubation, so it would not be surprising if the amount of resources dedicated to an egg is a fixed life history trait in this species.

Several additional results from this study are noteworthy. One, the variation of unmated female fecundity was much greater than the variation of mated female fecundity. Especially for the cumulative fecundity data (see Table 1), the variation of total egg number and total egg weight from unmated females was generally double the variation compared that of mated females. And two, both egg number and egg weight tended to decrease of their lifetime, and this was especially pronounced for unmated females (see Table 2). For example, while mated females were still producing an average of 25 10 THE TEXAS JOURNAL OF SCIENCE-VOL. 65, NO. 1, 2013 eggs over days 31 -40 and 41-50, unmated females were down to ~20 eggs over those same two time periods. Not surprisingly, this overall decrease in egg number over time is consistent with the cumulative fecundity data for the two treatment groups.

Four additional questions remain unanswered and provide a wealth of future research with M. dentricus. First, what is the ploidy of their eggs? Do unmated females lay haploid and/or diploid eggs? Many phasmid species have shown variation with respect to their ploidy (Pijnacker 1967; 1969; Marescalchi and Scali 1990; Giorgi 1992; Sandoval et al. 1998; Law and Crespi 2002; Scali et al. 2003; Ghiselli et al. 2007; Trewick et al. 2008; Schwander and Crespi 2008), and it would be interesting to understand M. dentricus on this proximate meiotic level. Second, how do considerations of co­ evolution affect the offspring from both mated and unmated females? (e.g., the Red Queen Hypothesis). In the long-term, clonal and/or haploid populations may be unable to escape organisms such as parasites that have specialized on their single genotype. Only studies that address multiple generations outside of the laboratory setting will allow us to understand the effects of parasites, predators, and other selective pressures related to co-evolutionary genetic phenomena. Third, how quickly do asexually reproducing sub­ populations acquire deleterious mutations? A study by Henry et al. (2012) found that asexually reproducing female stick insects had three to 13 fold higher rates of mutations in a suite of three . That is, for both nuclear and mitochondrial genes the rate of deleterious accumulation was higher in asexual populations compared to sexual (Henry et al. 2012). Whether M. dentricus experiences similar genetic effects is currently unknown but would prove insightful for the long-term fitness possibilities of wild, clonal populations. And fourth, does the hatching success of eggs from mated and unmated females differ? Hobbyists who rear these animals for recreation have kept female only populations for several years, so eggs from unmated females are certainly viable (Maginnis, personal communication). Given that egg weight (a possible predictor of hatching rate) appears to be a very consistent life history trait in M. MAGINN1S & REDMOND 11 dentricus, we hypothesize the hatching rates of the eggs produced by mated and unmated females are similar. This final unanswered question in M. dentricus is particularly important to understanding the evolutionary fitness costs of sexual versus asexual reproduction as Maynard Smith’s “cost of males” only applies if the offspring produced by asexual reproduction have the same fertility/ hatchability as the offspring produced by sexual reproduction (Bergerard 1958).

In conclusion, the results from this study suggest that the lifetime fecundity in unmated females is about 5-10% less than mated females. However, these differences are driven by the numbers of eggs laid relatively late in their reproductive period. During the first twenty days, there were no differences between the total numbers of eggs laid or the weight of those eggs, providing no support for the “cost of males” hypothesis. In fact, these results may be the most biologically relevant, as in natural populations only a subset of individuals will survive and continue to reproduce after two or three weeks. Future work that considers ecology as well as will help our understanding of the long-term fitness consequences of each reproductive strategy.

A cknowledgements We gratefully thank Andrea Pavia at St. Edward’s University for assistance in data collection and an anonymous reviewer for helpful comments on this manuscript. This manuscript is dedicated to the late Dr. Allan Hook, one of the finest entomologists to set foot in Texas.

L it e r a t u r e C it e d

Arnett. R. H. 2000. American Insects: a Handbook of the Insects of America North of Mexico. Second Edition. CRC Press. Boca Raton, Florida, 1024 pp. Bell, G. 1982. The Masterpiece of Nature: The Evolution of Genetics and Sexuality. Croom Helm, London. 643 pp. Bely, A. E. & K. G. Nyberg. 2010. Evolution of animal regeneration: re-emergence of a field. T.R.E.E. 25(3): 161—170. Bergerard, J. 1958. La parthenogenese des phasmids. A. Caracteres generaux. Bull. Biol. Fr. Belg. 92:90-102. 12 THE TEXAS JOURNAL OF SC1ENCE-VOL. 65, NO. 1, 2013

Brock. P. D. 1999. The Amazing World of Stick and Insects. AES Publishing. UK. 182 pp. Caudell A. N. 1903. The Phasmidae. or Walkingsticks, of the United States. Proc. U.S. Natl. Mus. 26:863-885. Crow, J. F. 1994. Advantages of sexual reproduction. Dev. Gen. 15:205-213. Enghoff, H. 1976. and bisexuality in the millipede Nemasoma varicorne (Diplopoda: Blaniulidae) Morphological, ecological, and biogeographical aspects. Videnskabelige Meddelelser fra Dansk Natuhistorike Forenening Kjfobenhavn 139:21-59. Felsenstein. J. 1974. The Evolutionary advantage of recombination. Genetics 78:737-756. Ghiselli, F., L. Milani. V. Scab & M. Passamonti. 2007. The Leptynia hispanica species complex (Insecta Phasmida): , parthenogenesis, hybridization and more. Mol. Ecol. 16:4256-4268. Giorgi. P. P. 1992. Sex and the male stick insect. Nature 357:444-445. Hamilton. W. D., R. Exalrod & R. Tanese. 1990. Sexual reproduction as an to resist parasites (a review). Proc. Nat. Acad. Sci. USA 87:3566-3573. Henry. L., T. Schwander & B. J. Crespi. 2012. Deleterious mutation accumulation in asexual Timema stick insects. Mol. Biol. Evol. 29( 1 ):401 —408. Kearney. M. & R. Shine. 2005. Lower fecundity in parthenogenetic geckos than sexual relatives in the Australian arid zone. J. Evol. Biol. 18:609-618. Lamb. R. Y. & R. B. Willey. 1979. Are parthenogenetic and related bisexual insects equal in fertility? Evol. 33(2):774-775. Law, J. H. & B. J. Crespi. 2002. The evolution of geographic parthenogenesis in Timema walking-sticks. Mol. Ecol. 11:1471—1489. Lloyd. D. G. 1980. Benefits and handicaps of sexual reproduction. Evol. Biol. 13:69-111. Lynch. M. 1984. Destabilizing hybridization, general-purpose genotypes and geographic parthenogenesis. Q. Rev. Biol. 59:257-290. Maginnis, T. L. 2006. Costs of regeneration and autotomy in animals: a review and framework for future research. Beh. Ecol. 17:857-872. Maginnis, T. L.. C. Cool & J. Muniz. 2008. Some observations on the mating behavior of the Giant Walking Stick, Megaphasma denlricus (Orthoptera: Phasmidae). TX. J. Sci. 59:1-6. Marescalchi. O. & V. Scab. 1990. Cytogenetic studies on Bacillus grandii grandii and B.grandii benazzii (Insecta. Phasmatodea): karyotype description, constitutive heterochromatin and nucleolus organizer regions. Genetica 82:117-124. Maynard Smith, J. 1971. What use is sex? J. Theor. Biol. 30:319-335. Maynard Smith. J. 1978. The Evolution of Sex. Cambridge University Press, Cambridge, 236 pp. Melian, C. J.. D. Alonso, S. Allesina, R. S. Condit. and R. S. Etienne. 2012. Does sex speed up evolutionary rate and increase ? PLoS Comput. Biol. 8(3):el 002414. Michod, R. E & B. R. Levin. 1988. The Evolution of Sex. Sinauer Press, Sunderland, MA. 342 pp. Misevic, D.. R. D. Kouyos & S. Bonfoeffer. 2009. Predicting the evolution of sex on complex fitness landscapes. PLoS Comput. Biol. 5(9):e 1000510. Muller, H. J. 1932. Some genetic aspects of sex. Am. Nat. 66(703): 118-138. MAGINN1S & REDMOND 13

Muller, H. J. 1964. The relation of recombination to mutational advance. Mutat. Res. 106:2-9. Otte, D. & P. D. Brock. 2005. Phasmida Species File: Catalog of stick and leaf insects of the world. The Insect Diversity Association and the Academy of Natural Sciences, Philadelphia, Cafe Press, 414 pp. Otto, S. P. & T. Lenormand. 2002. Resolving the paradox of sex and recombination. Nat. Rev. Genet. 3:252-261. Pijnacker, L. P. 1967. Oogenesis in the parthenogenetic stick insect Sipyloidea sipylus Westwood (Orthoptera, Phasmidae). Genetica 38:504-515. Pijnacker, L. P. 1969. Automictic parthenogenesis in the stick insect Bacillus rossius rossi (Cheleutoptera, Phasmidae). Genetica 40:393-399. Ridley, M. 1995. The Red Queen: Sex and the Evolution of Nature. New York. NY, 416 pp. Roze, D. 2012. Disentangling the benefits of sex. PLoS Biol. 10(5):e 1001321. Sandoval, C., D. A. Carmean & B. J. Crespi. 1998. Molecular of sexual and parthenogenetic Timema walking-sticks. Proc. Roy. Soc. B, 265:589-595. Scab, V., M. Passamonti, O. Mareschalchi & B. Mantovani. 2003. Linkage between sexual and asexual lineages: in Bacillus stick insects. Biol. J. Linn. Soc. 79:137-150. Schwander, T. & B. J. Crespi. 2008. Multiple direct transitions from sexual reproduction to apomictic parthenogenesis in Timema stick insects. Evol. 63:84-103. Song, Y., S. Scheu & B. Drossel. 2012. The ecological advantage of sexual reproduction in multicellular long-lived organisms. Evol. Biol. 25:556-565. Soumalainen, E. 1962. Significance of parthenogenesis in the . Ann. Rev. Ento. 7:349-366. Stenseth, N. C. 1979. Where have All the species gone? On the nature of extinction and the Red Queen hypothesis. Oikos 33(2): 196-227. Trewick, S. A., M. Morgan-Richards & L. J. Collins. 2008. Are you my mother? Phylogenetic analysis reveals orphan stick insect genus is part of a monophyletic New Zealand clade. Mol. Phylo. Evol. 48:799-808. Uzzel, T. M. 1964. Relations of the diploid and triploid species of Amystoma jeffersonianum complex (Amphibia, Caudata). Copeia 2:257-300. Van Valen, L. 1973. A new evolutionary law. Evol. Theory. 1:1-30. Weinzierl, R. P., P. Schmidt & N. K. Michiels. 1999. High fecundity and low fertility in parthenogenetic planarians. Invertebr. Biol. 118:87-94. Williams, G. C. 1975. Sex and Evolution. Princeton University Press, Princeton, NJ. 200 pp.

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