Evolution, 58(7), 2004, pp. 1511±1520

PARASITES AND SEXUAL REPRODUCTION IN PSYCHID

TOMI KUMPULAINEN,1,2 ALESSANDRO GRAPPUTO,1,3 AND JOHANNA MAPPES1,4 1Department of Biological and Environmental Science, PO Box 35, University of JyvaÈskylaÈ, FIN-40014 Finland 2E-mail: [email protected].® 3E-mail: [email protected].® 4E-mail: [email protected].®

Abstract. Persistence of sexual reproduction among coexisting asexual competitors has been a major paradox in evolutionary biology. The number of empirical studies is still very limited, as few systems with coexisting sexual and strictly asexual lineages have been found. We studied the ecological mechanisms behind the simultaneous coexistence of a sexually and an asexually reproducing closely related of psychid in Central Finland between 1999 and 2001. The two species compete for the same resources and are often infected by the same hymenopteran parasitoids. They are extremely morphologically and behaviorally similar and can be separated only by their reproductive strategy (sexual vs. asexual) or by genetic markers. We compared the life-history traits of these species in two locations where they coexist to test predictions of the cost-of-sex hypothesis. We did not ®nd any difference in female size, number of larvae, or offspring survival between the sexuals and asexuals, indicating that sexuals are subject to cost of sex. We also used genetic markers to check and exclude the possibility of Wolbachia bacteria infection inducing parthe- nogenesis. None of the samples was infected by Wolbachia and, thus, it is unlikely that these bacteria could affect our results. We sampled 38 locations to study the prevalence of parasitoids and the moths' reproductive strategy. We found a strong positive correlation between prevalence of sexual reproduction and prevalence of parasitoids. In locations where parasitoids are rare asexuals exist in high densities, whereas in locations with a high parasitoid load the sexual species was dominant. Spatial distribution alone does not explain the results. We suggest that the parasite hypothesis for sex may offer an explanation for the persistence of sexual moths in this system.

Key words. Bag worm moth, cost of sex, parasite hypothesis, parthenogenesis, Red Queen hypothesis, Wolbachia.

Received October 8, 2003. Accepted February 9, 2004.

Maintenance of sexual reproduction has been a puzzling uals, but in unfavorable environmental conditions, the situ- question in the theory of evolution since it was ®rst raised ation was reversed. by Williams (1975), Maynard Smith (1978), and Bell (1982). Some recent studies have focused on the mechanisms re- Many hypotheses have been presented to explain the main- stricting the success of facultative parthenogenesis within tenance of sex (e.g., Kondrashov 1993; Hurst and Peck 1996; species capable of both sexual and asexual reproduction, with Lively 1996; Jokela et al. 2003), since the asexually repro- the latter often functioning as a purely secondary reproduc- ducing morphs could produce offspring more effectively than tive strategy in the absence of a suitable mate (e.g., Corley the sexuals by avoiding the costs of producing males. This et al. 2001). Theoretical studies have often focused on the cost of sex should ®nally lead to replacement of sexuals if success of sexuals versus asexuals (e.g., Rispe and Pierre the all-else-equal assumption is true (Maynard Smith 1978). 1998; Doncaster et al. 2000; Peck and Waxman 2000), but In the long term, this should lead to natural populations con- the actual paradox of the maintenance of sex still remains. sisting purely of asexually reproducing females, unless there Apart from the outstanding studies on New Zealand fresh- are some short-term advantages of sexual reproduction (e.g., water snails (Lively 1992; Fox et al. 1996; Jokela et al. 1997; Lively 1996). Such short-term advantages could arise from Howard and Lively 1998), there is very little empirical data the genetic recombination in cross-fertilization, which purges from natural systems where sexually and asexually repro- deleterious mutations and increases the amount of genetic ducing morphs of nonhybrid origin coexist and compete with variation (Muller 1964; Kondrashov 1988; Rice 2002). each other (snail, Campeloma limum: Johnson 2000; ¯at- The importance of variation in the host genotype was fur- worm, Schmidtea polychroa: Michiels et al. 2001). ther developed in the parasite hypothesis (Hamilton 1980), In many species, asexuality is, in fact, not a true which emphasizes the effects of coevolution of host and par- reproductive strategy but caused by a feminizing bacteria, asite. These coevolutionary interactions, together with strong which can increase the mortality of male embryos. Some frequency-dependent selection by high parasite prevalence, strains of Wolbachia bacteria force genotypic males to act as could favor variability in host genotypes, and thus rare ge- phenotypic females and drive sexual females to reproduce notypes might avoid infections because parasites are most parthenogenetically (Bourtzis and O'Neill 1998). However, likely to track the most common genotypes (Hamilton 1980; these effects may be considered as true costs of producing Ooi and Yahara 1999; Lively and Dybdahl 2000). Such co- males as the asexually reproducing morphs rarely carry the evolution can lead to continuous changes in the frequency harmful effects of these bacterial infections (Bourtzis and of host genotypes. This idea is manifested Red Queen hy- O'Neill 1998; Kageyama et al. 1998). pothesis (Van Valen 1973) that has recently become widely Data on natural systems in which the effects of parasites studied. Support for the parasite hypothesis for sex was re- on their hosts are extremely severe is even more limited. cently offered by Negovetic and Jokela (2001), who found While some authors argue that it could be parasites or path- that asexual snails reproduced more successfully than sex- ogens causing serious reduction in the ®tness of their hosts 1511 ᭧ 2004 The Society for the Study of Evolution. All rights reserved. 1512 TOMI KUMPULAINEN ET AL. together with moderate rates of mutation that would most to investigate this hypothesis, the two species should coexist, likely favor sexual reproduction (e.g., Howard and Lively compete, and be infected by the same parasites. 1994, 1998), studies have been made mainly with less harm- ful parasites (e.g., Hanley et al. 1995; Michiels et al. 2001). MATERIALS AND METHODS More empirical data to support any of the hypotheses around Study System the maintenance of sex are clearly needed. Bag worm moths (: Psychidae) provide a good In Europe Siederia and Dahlica species (Lepidoptera: Psy- opportunity to investigate the coexistence of sexual and asex- chidae) are common, and many species have a wide distri- ual reproduction in the same location. In Lepidoptera, par- bution ranging from central Europe to northern Scandinavia. thenogenetic reproduction is very rare. However, in the fam- However, the status of many species is still uncertain, and ily Psychidae and especially among Dahlica species, parthe- species determination is mainly based on a few morpholog- nogenesis seems to have evolved several times (T. Kumpu- ical features (see Suomalainen 1980; HaÈttenschwiler 1997; lainen, A. Grapputo, and J. Mappes, unpubl. ms.), and thus Kumpulainen et al., unpubl. ms.). At least three separate par- several species with strictly parthenogenetic reproduction ex- thenogenetic species have been identi®ed in the subfamily ist in this group (see HaÈttenschwiler 1997). Many of these Solenobinae, and all appear to be tetraploids, probably orig- species occur in Finland and often coexist in the same habitats inating from different sexual species in the same subfamily with sexual species. The taxonomic relationships of this (Suomalainen 1980; HaÈttenschwiler 1997; Kumpulainen et group are still unclear and poorly studied. Previously, all al., unpubl. ms.). Although the origin of parthenogenesis in Dahlica and Siederia moths were classi®ed in the So- Dahlica and Siederia species is not known, it is unlikely to lenobia. So far, taxonomic classi®cation of these two genera be interspeci®c hybridization. A close relative, the parthe- has been based on morphological characters (the length of a nogenetic D. triquetrella, reproduces by automictic parthe- small spine in male front leg; see HaÈttenschwiler 1997). Re- nogenesis. In this type of parthenogenesis the zygoid phase cently, we investigated the phylogenetic relationships of is restored by the fusion of the two central nuclei, which these species based on mitochondrial DNA markers (T. Kum- results in the formation of the Richtungs-Kopulations-Kern pulainen, A. Grapputo, and J. Mappes, unpubl. ms.) and (RKK) structure. The fusion leads to heterogamy, which ex- found that sexually reproducing S. rupicolella and asexual plains why only females are produced. The RKK is also D. fennicella are closely related but clearly different species formed in the sexual form of D. triquetrella but is not de- (Suomalainen 1980). According to our molecular data, the veloped further and the zygoid phase is restored through the presence of these two genera is still questionable. Here we introduction of the sperm (see Suomalainen 1962). Many use current classi®cation to indicate the two species. Dahlica and Siederia species are not only morphologically Apart from the reproductive mode, we could not ®nd any uniform, but also share most of their ecological and biogeo- reliable differences in morphology, ecology, or behavior be- graphical features. In Finland, they occur mostly in older tween the sexual and asexual forms. In central Finland asex- wooded habitats, especially in sparse forests or warmer forest ual D. fennicella and sexual S. rupicolella coexist in many edges. Most species occur patchily, and the same habitats are locations. Some locations have only sexually reproducing often occupied by several species. species and others have only asexuals, but in most of the Bag worm moth larvae always carry a case (or bag) made locations both reproductive strategies coexist. The coexis- of plant material, sand, and other dry matter. Larvae feed on tence of closely related sexual and asexual moths and the moss and algae and ®nally pupate within their larval case. extremely heavy selective pressure caused by lethal hyme- Females of these species are wingless, sessile, and incapable nopteran parasitoids offers a good system to compare the of dispersal. Once hatched, sexual females excrete phero- competitive abilities of the reproductive groups, as well as mones to attract males. After copulation, females lay their to study the possible ecological factors behind the coexisting eggs in their own larval case and die within few hours after strategies. egg laying. Parthenogenetic females, instead, do not excrete The purpose of this study is to obtain much needed em- pheromones but start to lay eggs shortly after hatching. Males pirical data of coexisting and competing sexuals and asexuals are always winged and search for newly hatched females, as well as to scrutinize the two important predictions gen- guided by female pheromones, but males have very limited erally linked with the maintenance of sex. First, we studied dispersing ability and can only ¯y for short distances (10± whether the sexually reproducing group has costs from pro- 100 m). The length of an adult female of both species is on ducing males compared to the strictly asexual reproduction average 3±5 mm and the wingspan of male on average 13± in the parthenogenetic group, as the cost-of-sex hypothesis 16 mm. Larvae are about the size of a female and the larval suggests (Maynard Smith 1978). There is also the possibility case averages 6±8.5 mm. Life cycle from egg to adult takes of sexually transmitted bacteria causing lower survival in from one to two years, but the adult stage takes only three sexually reproducing individuals and their offspring (Bourtz- to six days (Suomalainen 1980). Hence, a moth spends most is and O'Neill 1998). Thus, we tested for Wolbachia bacteria of its life cycle as a larva. to rule out the possibility of parthenogenesis or other repro- Psychid larvae are often infected by at least two common ductive features being in¯uenced by this bacteria. Second, species of hymenopteran parasitoids (see HaÈttenschwiler we examined whether strong selective pressures such as high 1997; P. HaÈttenschwiler, pers. comm.), including Orthizema parasitoid prevalence could favor sexually reproducing spe- spp. and a few rare species (Hymenoptera: Ichneumonidae). cies, which is the basic assumption of the parasite hypothesis In all parasitoid species, females normally lay one egg per for sex (e.g., Hamilton 1980; Jokela et al. 2003). To be able host larva. The parasitoid egg remains dormant until the fully PARASITES AND SEXUAL REPRODUCTION 1513

FIG. 1. Map of Finland. (A) Area of the northern populations, locations 34±37; (B) the main study area in Central Finland, locations 1±27, 41±48; (C) area of the southern populations, locations 28±33. grown host larva pupates, when the parasitoid larva quickly Appendix). Both asexuals and sexuals are abundant in these eats the host, pupates, and hatches just a few weeks after the locations. Study areas consisted of older forest patches, sep- moth's hatching period. During larval development, these arated by lakes and ®elds and sometimes by human settle- hymenopteran parasitoids kill their hostsÐhence the cost of ments. Forests are dominated by Norwegian spruce (Picea being parasitized is extreme. Because the infected individuals abies) and silver birch (Betula pendula), many of them also are always lost, their reproductive strategy or species cannot containing Scots pine (Pinus sylvestris) and occasionally a be determined. In preliminary laboratory experiments, we few European aspen (Populus tremula) or grey alder (Alnus offered different host larvae to females of the most common incana). Individuals were collected in April of 1999, 2000, parasitoid species. The parasitoids did not discriminate be- and 2001. To catch ®nal instar larvae, which climb on tree tween host larvae of different Dahlica or Siederia species or trunks to pupate in early spring, we set tape traps on tree between larvae of different size, as long as the larvae were trunks where larvae get stuck and can be later collected. At fully grown (i.e., the larval case was at least 6 mm long). On Sip 1 and Huvin, we set 138 and 83 tape traps, respectively the basis of previous observations, we knew that both the (see Appendix). The traps were set before the beginning of parasitoid prevalence and the proportion of sexuals versus the climbing period (30 March to 7 April), and they were asexuals vary strongly between the locations under study. S. checked twice per week until the climbing period was over rupicolella (sexual) and D. fennicella (asexual) are locally (30 April). Each larva was separately labeled and taken into the most common species (80% or more of all species of the laboratory for further studies. psychid moths). In the laboratory, all collected larvae were kept individ- ually in transparent plastic containers (height 6.7 cm; di- Cost of Sex ameter 3.7 cm) with foam lids to allow enough oxygen to Individuals for the laboratory studies of cost of sex were pass. Temperature and humidity were kept at outdoor levels. collected from two locations in 1999, Sip 1 and Huvin, in The larvae pupated soon after collection and, about three JyvaÈskylaÈ, Central Finland (62Њ15ЈN, 25Њ43ЈE; see Fig. 1, weeks later, males, sexual females, and asexual females be- 1514 TOMI KUMPULAINEN ET AL. gan to hatch. The reproductive strategy of female moths was 400 ␮M of each dNTPs, and 0.5 units of Taq polymerase determined on the basis of female behavior, that is, whether (Gibco-BRL, Gaithersburg, MD). PCR cycling conditions the females waited for males or started to lay eggs without were one cycle at 94ЊC for 5 min, followed by 30 cycles of copulation. After hatching, all females climbed on the top of 30 sec at 94ЊC, 30 sec at 50ЊC, and 1 min at 72ЊC followed their own larval case. Parthenogenetic females immediately by one cycle at 72ЊC for 5 min. The PCR products were started to lay eggs inside their larval case, whereas sexual resolved in a 1% agarose gel stained with ethidium bromide. females began to release pheromones to attract males and did Naturally infected ants, Gnamptogenys menadensis, were not start egg laying without copulation. used as a positive control, and uninfected ants, G. moelleri, We offered a randomly chosen male to each sexual female. were used as a negative control in each ampli®cation. To Males quickly began ¯ying toward the female and copulation check if DNA extraction was successful, PCR was also pre- normally succeeded immediately and lasted between 5 and formed using primers S2792 and A3389 (Simon et al. 1994), 30 min. After copulation, sexual females laid their eggs inside which amplify a fragment of the mitochondrial COII gene. their own larval case. Because females die within few hours of egg laying, they are only able to copulate and reproduce Parasitoid Prevalence and Proportion of Sexual once. Egg clutches laid by individual females were kept sep- Reproduction arate. The number and proportion of hatching larvae were calculated. Samples for parasitoid prevalence studies were collected After egg-laying, sexual and asexual females from Huvin from similar habitats as the moths for the cost-of-sex studies (location 3) and Sip 1 (location 1) were individually weighed (see above). However, prevalence of parasitoids in the study to the nearest 0.1 mg using a HA-202M microbalance (A and species in 1999 was determined in 15 different study loca- D Instruments, Oxford, U.K.) We measured the length of tions within a relatively small area (15 km ϫ 30 km; see females, pupae, and larval cases. Of all female measurements, Appendix) around JyvaÈskylaÈ, Central Finland (62Њ15ЈN, the pupal length proved to be the most reliable measurement 25Њ43ЈE: see Fig. 1). In the following two years, 2000 and of female size, and thus it was used to indicate female size 2001, the parasitoid prevalence and the proportion of sexual in our study. reproduction were determined in 29 and 19 locations, re- spectively (for reproductive strategy determination, see the F1 clutches produced by sexual and asexual females were grown in laboratory conditions (19 Ϯ 2ЊC, 80% humidity, cost-of-sex discussion above). In 2000, we also sampled ad- food ad libidum) similar to their natural habitats. The survival ditional areas in southern Finland, near Orimattila, and in the rates of moth larvae were checked before the overwintering northern part of Central Finland, around SonkajaÈrvi (see Ap- period. Because females of sexually and asexually reproduc- pendix and Fig. 1 for exact locations). In each area, we set ing species are practically impossible to separate reliably ®ve to 138 tape traps (see Appendix). In smaller forest patch- before their reproduction mode, we were forced to randomly es, practically all trees were tape trapped, whereas in larger collect as many individuals per location as possible. Because forest patches trunks were chosen randomly. Location 4 (Isa) the adults are very short lived (maximum three to six days) was exceptional in 1999, since all bag worms of this popu- and only males hatching on the same day as females could lation were collected from the walls of a house and, thus, be successfully mated, there were only a few, if any, suitable they were hand picked. Sample sizes are presented in the mate choices at a time; therefore, sample sizes of reproductive Appendix. success for sexuals remained lower than for asexuals. Although some of the study locations are close to each other (in certain cases less than 200 m), we treated them as separate populations since preliminary genetic data of an on- Wolbachia Analysis going study (A. Grapputo, T. Kumpulainen, and J. Mappes, Total genomic DNA was extracted from whole single adult unpubl. ms.) shows very low gene ¯ow between neighboring females of sexual S. rupicolella, parthenogenetic D. fenni- populations. Moreover, females do not disperse and males cella, and their hymenopteran parasitoids in 1999 using the can ¯y only very short distances. In our study system, re- Chelex extraction method (Walsh et al. 1991). Eighty-®ve productive strategy results are based on moth individuals not females (44 parthenogenetic and 41 sexual) and 10 parasit- infected by parasitoids, as infected individuals are always oids were tested for the presence of the bacteria Wolbachia lost and neither their reproductive strategy nor their species pipientis via polymerase chain reaction (PCR). These samples can be determined. were collected randomly from several study populations. In the laboratory, reproductive mode and sex of the hatch- Three sets of primers were used to amplify two Wolbachia ing moths was monitored. Approximately four weeks after genes, wsp (the gene encoding the major surface protein) and the adult moths' hatching period (two months after collecting 16S rDNA. The general primers wsp 81F and 691R (Braig the larvae), the parasitoids started to hatch from the moth et al. 1998), which are able to amplify both major strains of pupa, which had been infected in the ®eld. The parasitoid Wolbachia (A and B), were used to amplify approximately prevalence in all locations was determined and compared to 600 bp of the wsp gene. The primers 16SAf ϩ 16SAr and the proportions of sexually and asexually reproducing moths 16SBf ϩ 16SBr, speci®c to the Wolbachia strains A and B, in each location. Our study species are infected by several respectively, were used to amplify approximately 250 bp of species of Hymenopteran parasitoids belonging mostly to ge- the 3Ј portion of the 16S rDNA. nus Orthizema. Here the parasitoid prevalence means total PCR was performed in a total volume of 25 ␮l using 10 parasitoid load and thus covers all the hymenopteran para- mM of Tris-HCl, 1.5 mM MgCl2, 5 pmoles of each primer, sitoids hatching from the collected host moths. The effects PARASITES AND SEXUAL REPRODUCTION 1515

parasitoids (rs1999 ϭ 0.802, N ϭ 15, P Ͻ 0.001; rs2000 ϭ 0.542, N ϭ 29, P ϭ 0.002; rs2001 ϭ 0.811, N ϭ 19, P Ͻ 0.001). This may suggest that sexual reproduction could com- pensate for the costs of sex in environments with heavy par- asitoid loads (Fig. 3). Although sexuality/asexuality seems to be strongly clustered, geographic distribution does not explain the occurrence of the reproductive strategies (Man- tel's test: r1999 ϭ 0.04, P ϭ 0.712; r2000 ϭ 0.05, P ϭ 0.554; r2001 ϭ 0.11, P ϭ 0.094). The size of the habitat patches did not correlate with prevalence of parasitoids in any year (all P Ͼ 0.600). The density of populations (individuals per trap) varied from 0.1 to 34 among locations. There was a strong correlation in 2000 (rs2000 ϭϪ0.64, N ϭ 29, P Ͻ 0.001) and FIG. 2. The number of larvae at hatching (black bars) and surviving a nonsigni®cant tendency in 1999 (rs1999 ϭϪ0.52, N ϭ 14, larvae after one month (gray bars) and in fall (white bars). Vertical P ϭ 0.056) for prevalence of parasitoids to correlate nega- lines show standard deviations. tively with host density. In 2001, with the smallest sample size, there was no correlation (rs2001 ϭϪ0.045, N ϭ 13, P ϭ 0.170). This indicates that in locations with high parasitoid of habitat characteristics (size, location, and main tree spe- loads host populations are sparse. The proportion of sexuals cies) on the proportion of sexual reproduction in each location did not correlate with population density (r 0.29, N were also analyzed. s1999 ϭϪ ϭ 14, P ϭ 0.300; rs2000 ϭϪ0.157, N ϭ 29, P ϭ 0.416; rs2001 ϭϪ0.44, N ϭ 12, P ϭ 0.148). To maintain reliability of RESULTS these analyses, sample sizes smaller than 10 individuals per Cost of Sex location were excluded from the Spearman correlations when analyzing proportion of sexuality in locations. Comparison of pupal length measurements did not reveal any signi®cant differences in size between the females of sexual S. rupicolella and asexual D. fennicella (two-way DISCUSSION ANOVA for ranked data: F1,358 ϭ 0.567, P ϭ 0.452) or We found that sexual and asexual females have equal re- between the two locations (F1,358 ϭ 0.855, P ϭ 0.356). We productive output, which indicates that sexuals may suffer did not ®nd any signi®cant differences between species in from the cost of sex (Maynard Smith 1978) as half of their the number of hatched larvae nor in the number of larvae offspring will be males (see Fig. 2). that survived until the following autumn (ANOVA for Most interestingly, we found a strong positive correlation ranked data: F1,358 ϭ 0.017, P ϭ 0.897; F1,358 ϭ 0.215, P between parasitoid prevalence and the occurrence of sexual ϭ 0.643) or between locations (F1,358 ϭ 0.904, P ϭ 0.342; reproduction in three years (Fig. 3). This means that sexually F ϭ 3.40, P ϭ 0.066; see Fig. 2). Sexuals had only 1,358 reproducing moths (S. rupicolella) are usually abundant in 0.37% higher reproductive output (number of hatched lar- locations where lethal hymenopteran parasitoids are com- vae) at Sip 1 (location 1) when compared to asexuals. At mon, while asexuals (D. fennicella) dominate where the par- Huvin (location 3) asexuals had 3.2% higher reproductive asitoid prevalence is lower. However, due to the nature of output (Fig. 2). However, we found a signi®cant difference in hatching date between asexuals and sexuals. Asexuals our study system, we are not able to compare the reproductive tended to hatch on average ®ve days earlier than sexuals at strategy of infected versus noninfected moths because the Sip 1 and six days earlier at Huvin (Mann-Whitney U-test, infected individuals are always lost and their reproductive Z ϭ 2.4, n ϭ 10, n ϭ 263, P ϭ 0.018; Z ϭ 3.2, n ϭ 10, strategy or species therefore cannot be determined. Thus, our 1 2 1 results on reproductive strategy are based on moth individuals n2 ϭ 83, P ϭ 0.002, respectively). uninfected by parasitoids. Wolbachia In recent publications, the foremost hypothesis to explain the evolution of sex is the parasite hypothesis for maintenance No infection from W. pipientis was detected in females of of sex, which relies on coevolutionary interactions between either species. None of the genes (wsp and 16S rDNA) were host and parasite (Hamilton 1980) and particularly on genetic detected by ampli®cation, whereas 90% of the parasitoids variability in resistance to the parasite (e.g., Grosholz 1994). were infected with both A and B strains of Wolbachia. The parasite hypothesis states that coevolutionary interac- tions between host and parasite, together with strong fre- Parasitoid Prevalence and Proportion of Sexual quency-dependent selection caused by the parasites, could Reproduction favor variability in host genotypes, because parasites are most The proportion of sexually reproducing females (of all likely to track the most common genotypes and the rarer hatched females) varied from 0% to 100% among the 38 study genotypes may avoid parasite infection (see Hamilton 1980). locations. The proportion of sexually reproducing females in Thus, the occurrence of common parasites could favor sex- these locations was signi®cantly and positively correlated uals with a higher recombination rate unless the number of with the proportion of moth larvae infected by hymenopteran asexual clones is very high (for discussion about the hy- 1516 TOMI KUMPULAINEN ET AL.

by the common hymenopteran parasitoids (HaÈttenschwiler 1997; P. HaÈttenschwiler, pers. comm.). Moreover, our own ®eld and laboratory observations (1999±2002) indicate that parasitoids do not discriminate between the larvae of different host species as long as the larvae are fully grown and have not been previously infected by other parasitoids. The general value of the parasite hypothesis has been high- lighted by previous studies on at least two different fresh- water snail species (Lively 1987, 1992; Johnson 2000), bi- valves (Grosholz 1994), ¯atworms (Michiels et al. 2001), geckos (Moritz et al. 1991), ®sh (Lively et al. 1990), and yeast (Greig et al. 1998). In our study system, the evolu- tionary forces between the psychid moths and their parasit- oids may well give rise to an advantage for sexual repro- duction, if the coadapted hymenopteran parasitoids are able to infect the most common host genotypes and if the asexual hosts have less genetic variability than the sexuals (Hamilton 1980; Hurst and Peck 1996; Lively 1996). In such a situation, only a small proportion of rare or new, and thus most likely sexually produced, genotypes could escape the parasites or other strong selective pressure (see Greig et al. 1998). Thus, parasite pressure could favor sexual reproduction if such re- production produces more genetic variation in the population. In cases where the asexual population contains more genetic variation than the sexuals, parasite prevalence is not expected to favor sexuality (see Lively et al. 1990; Hanley et al. 1995). In general, lethal parasites are thought to have the most re- markable effects on competition between closely related sex- ual and asexual species (see May and Anderson 1983). More- over among psychid moths, remarkable effects on ®tness are present, as the infected individuals are excluded from the population before they mature. Many previous empirical studies with comparative per- spectives have been made between sexuals and parthenogens of hybrid origin (Vrijenhoek et al. 1989; Lively et al. 1990; Moritz et al. 1991; Radtkey et al. 1995; Schartl et al. 1995; Mantovani et al. 1996). In certain breeding systems inter- dependence between the reproductive strategies still exists. Among the gynogenetic ®sh species Poecilia, the partheno- genetic reproduction is dependent on the availability of males of the sexual competitors. Although the male's genome does not contribute to the next generation, the sperm is still needed to activate the development of the embryo (Hubbs and Hubbs 1932). In systems with asexual competitors of hybrid origin, comparison between the sexuals and asexuals may be dif®cult because the hybrids have new genetic combinations origi- nating from two different genomes and they are often sub- jected to outbreeding effects such as elevated heterozygocity levels (see Jokela et al. 1997). In our study, the parthenogenetic moth is most likely an independent species of nonhybrid origin (Kumpulainen et al., FIG. 3. Correlation between parasitoid prevalence and the pro- portion of sexuality (A) in 1999 at 15 locations; (B) in 2000 at 29 unpubl. ms.). According to our phylogenetic studies, S. rup- locations; and (C) in 2001 at 19 locations. Names and descriptions icolella and D. fennicella are close relatives and members of of the study locations are given in the Appendix. a compact phylogenetic group (Kumpulainen et al., unpubl. ms.). Previously, niche separation has been found also among nonhybrid clones of at least the moth Alsophila pometaria potheses, see Hamilton 1980; Lively 1992, 1996; Greig et (Futuyama et al. 1981; Harshman and Futuyama 1985) and al. 1998; Jokela et al. 2003). Ips bark beetles (Kirkendall and Stenseth 1990). However, Our result appears to support the parasite hypothesis, since in our system, we were not able to de®ne the possible niche both sexual and asexual moth species are frequently infected differences of the sexual and asexual moths. Furthermore, PARASITES AND SEXUAL REPRODUCTION 1517 both species were successfully grown under same laboratory such as temperature or other abiotic conditions that could be conditions and with same larval host plants. This, together unfavorable to both parasitoids and sexual reproduction in with all the information about the ecological and morpho- certain locations where asexuals manage to persist. Thus, our logical similarity of our study species (see Suomalainen system could be a case of geographical parthenogenesis, a 1980; HaÈttenschwiler 1997), gives us reasonable grounds to situation in which the sexual and the parthenogenetic rela- compare the success of asexual and sexual reproduction. tives differ in their distribution areas, often due to some en- If any cost of sex exists, the conditions for the all-else- vironmental conditions (Seiler 1923; Vandel 1928; Suoma- equal requirement should be met. In our study, we could not lainen 1950; Glesener and Tilman 1978). The logic behind ®nd any signi®cant differences in the reproductive output of geographical parthenogenesis is that asexuals do not have to sexual S. rupicolella and asexual D. fennicella females. If a mate and recombine their genome with nonoptimal mates parthenogenetic female is able to produce the same amount (possibly migrants), which might not have the necessary ad- of offspring as a pair of sexual moths, a cost of sexual re- aptations for the environment (Peck et al. 1998; see also production exists in sexual S. rupicolella. To our knowledge, Maynard Smith 1978; Greig et al. 1998). Both of our study this kind of cost for sexual reproduction in noncyclical species live in presumably harsh conditions, on the edges of (or other arthropod species) compared to coexisting asexual their distribution area (Suomalainen 1980). This is one reason species has not been detected in earlier empirical studies with why asexuals are expected to be better in colonizing new no indication of bacterial sex-ratio distortion. According to habitats and why they are expected to be found on the edges our results, no additional cost of sex caused by Wolbachia of the distribution area. However, we do not think that habitat exists here, and the causes of parthenogenesis in D. fennicella quality alone would explain our results. Neither habitat size are to be found elsewhere. (Appendix) nor geographical location had a signi®cant effect Many earlier studies found that the number of eggs pro- on the proportion of sexually reproducing moths in any of duced or survival at larval phases is lower in asexuals than the three study years (see Materials and Methods). Further- in sexuals (e.g., Bell 1982; Corley et al. 2001). In such sys- more, parasitoids are present to some extent in all study areas. tems, asexuality may function as a secondary reproductive The sexual and asexual moths coexist in most of the locations, strategy when sexual reproduction is not possible. In D. fen- and we did not ®nd indication that sexuals and asexuals would nicella, parthenogenesis is the sole reproductive strategy and be found preferentially in habitats that clearly differed in no males are known for this species. Facultative partheno- type, size, or structure (see Appendix). However, we cannot genesis is not known to exist among any solenobid psychids. totally exclude the possibility that some (ecological) factor It has been shown that, with a considerable rate of mu- could affect the apparent correlation between the parasitoid tations per genome (Keightley and Eyre-Walker 2000), mu- prevalence and sexual reproduction detected in this study. tation accumulation in the asexuals could explain why sex- Our results suggest a connection between the occurrence uals can outnumber the asexuals (Lively 1996; Peck and of sexually reproducing S. rupicolella and levels of parasit- Waxman 2000; Rice 2002). Even though the rate of mutation oids. This means that the hymenopteran parasitoids are likely per genome has not been studied in psychid moths, most to prevent the asexual D. fennicella from outnumbering their asexual populations are quite small (see Materials and Meth- sexual competitors in populations where the parasitoid prev- ods) and hence the conditions could also be suitable for Mull- alence is high. Without the parasitoids, the more effectively er's ratchet (Muller 1964) to work. However, West et al. reproducing asexuals should be able to displace their sexual (1999) emphasized the importance of a pluralistic view in- competitors. So, why have these parasitoids not infected all stead of ®nding simple explanations for the maintenance of the asexuals and caused their total extinction from their dis- sex in complex systems, and Howard and Lively (1998) sug- tribution area? There is no simple answer, but one possible gested that even moderate effects of parasites together with explanation may be hidden in the genetic variability of the mutation accumulation could act in favor of sex. However, competing species. Recently, we found that genetic vari- in our study system it is unlikely that mutation accumulation ability is higher in sexual species than in asexual species but could explain the maintenance of sexuals as the life-history clonal diversity in asexual D. fennicella is still very high (A. measurements do not indicate any phenotypic abnormalities Grapputo, T. Kumpulainen, and J. Mappes, unpubl. ms.). among the asexuals. Thus, it is likely that clonal extinction is relatively common One interesting ®nding in our study was that the highest and a replacement process of new clones frozen from sexual parasitoid prevalence was found in the most sparsely popu- populations maintains the asexual population (Vrijenhoek lated locations. This ®nding is quite opposite to the general 1979). expectations of the host-parasite interactions, where strong Our results give rare and needed empirical information of density dependence between parasitoids and hosts is expected a novel system where a sexually reproducing species main- (e.g., Begon et al. 1996). The reason for our ®ndings is un- tains itself among coexisting and more effectively reproduc- clear, but it is possible that in the densest asexual locations ing asexual competitors. Overall, this result shows that in prevalence of parasitoids would increase in following years. systems suitable for studying the evolution of sex, species However, in four locations where asexuals dominated in boundaries should not prevent the comparison of asexual and 1999, the parasitoid prevalence increased in following years, sexual reproduction if these reproductive morphs are of com- whereas in one location prevalence decreased and in one mon ancestry and still share most of their ecological features. location the prevalence remained the same (Fig. 3). In summary, our results indicate that there are costs of The observed correlation between proportion of sexuality reproducing sexually among Siederia moths and that these and parasitoid prevalence could result from other factors, costs are not caused by microorganisms such as Wolbachia. 1518 TOMI KUMPULAINEN ET AL.

Our results also indirectly suggest that the severe costs of tribution of clonal diversity in Alsophila pometaria (Lepidoptera: lethal parasites on asexual reproduction could help to main- Geometridae). Evolution 39:315±324. HaÈttenschwiler, P. 1997. Die SacktraÈger der Schweiz. Pp. 165±308 tain sex in populations of both reproductive groups. Thus, in Schweizerische Bund fuÈr NaturSchutz, Schmetterlinge und our main results are in concordance with cost-of-sex hy- ihre lebensraÈume, Arten-GefaÈhrung-Schutz. Band 2. Fotorotar, pothesis and with the parasite hypothesis for sex. Egg, Switzerland. Howard, R. S., and C. M. Lively. 1994. Parasitism, mutation ac- cumulation and the maintenance of sex. Nature 367:554±557. ACKNOWLEDGMENTS ÐÐÐ. 1998. The maintenance of sex by parasitism and mutation We sincerely thank K. Kulmala for his valuable help in accumulation under epistatic ®tness functions. Evolution 52: ®eldwork, laboratory rearing, and species determinations. We 604±610. Hubbs, C. L., and L. C. Hubbs. 1932. Apparent parthenogenesis in also thank E. Kiukkanen for maintaining conditions in the nature, in a form of ®sh of hybrid origin. Science 76:628±630. laboratory and P. Halme, V. Heino, T. Hiltunen, I. Kananen, Hurst, L. D., and J. R. Peck. 1996. Recent advances in understanding A. Kolehmainen, and L. Mattila for their help in the ®eldwork of the evolution and maintenance of sex. Trends Ecol. Evol. 11: and laboratory rearing. We also thank J. Jokela, K. E. Knott, 46±52. Johnson, S. G. 2000. Population structure, parasitism, and survi- M. Puurtinen, and the members of the round table meeting vorship of sexual and autodiploid parthenogenetic Campeloma for their very helpful comments on the manuscript. M. Iversen limum. Evolution 54:167±175. kindly commented on the language. Authors after ®rst author Jokela, J., C. M. Lively, M. F. Dybdahl, and J. A. Fox. 1997. are in alphabetical order. This study was done at the Uni- Evidence for a cost of sex in the freshwater snail Potamopyrgus versity of JyvaÈskylaÈ and it was ®nancially supported by the antipodarum. Ecology 78:452±460. ÐÐÐ. 2003. Genetic variation in sexual and clonal lineages of a Academy of Finland, project number 45812. freshwater snail. Biol. J. Linn. Soc. 79:165±181. Kageyama, D., S. Hoshizaki, and Y. Ishikawa. 1998. Female-biased LITERATURE CITED sex ratio in the Asian corn borer, Ostrinia furnacalis: evidence for the occurrence of feminizing bacteria in an . Heredity Begon, M., J. L. Harper, and C. R. Townsend. 1996. Parasitism and 81:311±316. disease. Pp. 429±481 in M. Begon, J. L. Harper and C. R. Town- Keightley, P. D., and A. Eyre-Walker. 2000. Deleterious mutations send. eds. Ecology: individuals, populations and communities. and the evolution of sex. Science 290:331±333. Blackwell, Oxford, U.K. Kirkendall, L. R., and N. C. Stenseth. 1990. Ecological and evo- Bell, G. 1982. The masterpiece of nature: the evolution and genetics lutionary stability of sperm-dependent parthenogenesis: effects of sexuality. Univ. of California Press, Berkeley, CA. of partial niche overlap between sexual and asexual females. Bourtzis, K., and S. O'Neill. 1998. Wolbachia infections and ar- Evolution 44:698±714. thropod reproduction: Wolbachia can cause cytoplasmic incom- Kondrashov, A. S. 1988. Deleterious mutations and the evolution patibility, parthenogenesis and feminisation in many . of sexual reproduction. Nature 336:435±440. Bioscience 48:287±293. ÐÐÐ. 1993. Classi®cation of hypotheses on the advantage of Braig, H. R., W. Zhou, S. L. Dobson, and S. L. O'Neill. 1998. amphimixis. J. Hered. 84:372±387. Cloning and characterization of a gene encoding the major sur- face protein of the bacterial endosymbiont Wolbachia pipientis. Lively, C. M. 1987. Evidence from a New Zealand snail for the J. Bacteriol. 180:2373±2378. maintenance of sex by parasitism. Nature 328:519±521. Buckling, A., and P. B. Rainey. 2002. The role of parasites in ÐÐÐ. 1992. Parthenogenesis in a freshwater snail: reproductive sympatric and allopatric host diversi®cation. Nature 420: assurance versus parasitic release. Evolution 46:907±913. 496±499. ÐÐÐ. 1996. Host-parasite coevolution and sex. Do interactions Corley, L. S., J. R. Blankenship, and A. J. Moore. 2001. Genetic between biological enemies maintain genetic variation and variation and sexual reproduction in the facultatively parthe- cross-fertilization? Bioscience 46:107±114. nogenetic cockroach Nauphoeta cinerea: implications for the Lively, C. M., and M. F. Dybdahl. 2000. Parasite adaptation to evolution of sex. J. Evol. Biol. 14:68±74. locally common host genotypes. Nature 405:679±681. Doncaster, C. P., G. E. Pound, and S. J. Cox. 2000. The ecological Lively, C. M., C. Craddock, and R. Vrijenhoek. 1990. Red Queen cost of sex. Nature 404:281±285. hypothesis supported by parasitism in sexual and clonal ®sh. Fox, J. A., M. F. Dybdahl, J. Jokela, and C. M. Lively. 1996. Genetic Nature 344:864±866. structure of coexisting sexual and clonal subpopulations in a Mantovani, B., F. Tinti, M. Barilani, and V. Scali. 1996. Current freshwater snail (Potamopyrgus antipodarum). Evolution 50: reproductive isolation between ancestors of natural hybrids in 1541±1548. Basillus stick insects (Insecta: Phasmatodea). Heredity 77: Futuyama, D. J., S. L. Leipertz, and C. Mitter. 1981. Selective 261±268. factors affecting clonal variation in the fall cankerworm Also- May, R. M., and R. M. Anderson. 1983. Epidemiology and genetics phila pometaria (Lepidoptera: Geometridae). Heredity 47: in the coevolution of parasites and hosts. Proc. R. Soc. Lond. 151±172. B 219:281±314. Glesener, R. R., and D. Tilman. 1978. Sexuality and the components Maynard Smith, J. 1978. The evolution of sex. Cambridge Univ. of environmental uncertainty: clues from geographical parthe- Press, Cambridge, U.K. nogenesis in terrestrial . Am. Nat. 112:659±673. Michiels, N. K., L. W. Beukeboom, N. Pongratz, and J. Zeitliger. Greig, D., R. H. Borts, and E. J. Louis. 1998. The effect of sex on 2001. Parthenogenetic ¯atworms have more symbionts than their adaptation to high temperature in heterozygous and homozygous coexisting, sexual conspesi®cs, but does this support the Red yeast. Proc. R. Soc. Lond. B 265:1017±1023. Queen? J. Evol. Biol. 14:110±119. Grosholz, E. D. 1994. The effects of host genotype and spatial Moritz, C., H. McCallum, S. Donnellan, and J. D. Roberts. 1991. distribution on trematode parasitism in a bivalve population. Parasite loads in parthenogenetic and sexual lizards support for Evolution 48:1514±1524. the Red Queen hypothesis. Proc. R. Soc. Lond. B 244:145±149. Hamilton, W. D. 1980. Sex versus non-sex versus parasite. Oikos Muller, H. J. 1964. The relation of recombination to mutational 35:282±290. advance. Mutat. Res. 1:2±9. Hanley, K. A., R. Fisher, and T. J. Case. 1995. Lower mite infes- Negovetic, S., and J. Jokela. 2001. Life-history variation, pheno- tations in an asexual gecko compared with its sexual ancestors. typic plasticity, and subpopulation structure in a freshwater snail. Evolution 49:418±426. Ecology 82:2805±2815. Harshman, L. G., and D. J. Futuyama. 1985. The origin and dis- Ooi, K., and T. Yahara. 1999. Genetic variation of geminiviruses: PARASITES AND SEXUAL REPRODUCTION 1519

comparison between sexual and asexual host plant populations. merase chain-reaction primers. Ann. Entomol. Soc. Am. 87: Mol. Ecol. 8:89±97. 651±701. Peck, J. R., and D. Waxman. 2000. Mutation and sex in a com- Suomalainen, E. 1950. Parthenogenesis in animals. Adv. Genet. 3: petitive world. Nature 406:399±404. 193±253. Peck, J. R., J. M. Yerslay, and D. Waxman. 1998. Explaining the ÐÐÐ. 1962. Signi®cance of parthenogenesis in the evolution of geographic distributions of sexual and asexual populations. Na- insects. Annu. Rev. Entomol. 7:349±365. ture 391:889±892. ÐÐÐ. 1980. The Solenobiinae species of Finland (Lepidoptera: Radtkey, R. R., S. C. Donnellan, R. N. Fisher, C. Moritz, K. A. Psychidae), with a description of a new species. Entomol. Scand. Hanley, and T. J. Case. 1995. When species collide: the origin 11:458±466. and spread of an asexual species of gecko. Proc. R. Soc. Lond. Vandel, A. 1928. La partheÂnogeneÁse geÂographique contribution a B. 259:142±152. leÂtude biologique et cytologique de la partheÂnogeneÁse naturelle. Bull. Biol. Fr. Belg. 62:164±281. Rice, W. R. 2002. Experimental tests of the adaptive signi®cance Van Valen, L. 1973. A new evolutionary law. Evol. Theory 1:1±30. of sexual recombination. Nat. Rev. Genet. 3:241±251. Vrijenhoek, R. C. 1979. Factors affecting clonal diversity and co- Rispe, C., and J.-S. Pierre. 1998. Coexistence between cyclical par- existence. Am. Zool. 19:787±797. thenogens, obligate parthenogens, and intermediates in a ¯uc- Vrijenhoek, R. C., R. M. Dawley, C. J. Cole, and J. P. Bogart. 1989. tuating environment. J. Theor. Biol. 195:97±110. A list of the known unisexual vertebrates. Pp. 24±31 in R. M. Schartl, M., B. Wilde, I. Schlupp, and J. Parzefall. 1995. Evolu- Dawley, and J. P. Bogart, eds. Evolution and ecology of uni- tionary origin of a parthenoform, the Amazon molly Poecilia sexual vertebrates. New York State Museum, Albany, NY. formosa, on the basis of a molecular genealogy. Evolution 49: Walsh, P. A., D. A. Metzger, and R. Higuchi. 1991. Chelex 100 as 827±835. a medium for simple extraction DNA for PCR-based typing from Seiler, J. 1923. Geschlechtschromosomenuntersuchungen an Psy- forensic material. BioTechniques 10:506±513. chiden. IV. Die parthenogenese der Psychiden. Z. induckt. Abs- West, S. A., C. M. Lively, and A. F. Read. 1999. A pluralist ap- tamm.-u. VerebLehre 31:1±99. proach to sex and recombination. J. Evol. Biol. 12:1003±1012. Simon, C., F. Frati, A. Beckenbach, B. Crespi, H. Liu, and P. Flook. Williams, G. C. 1975. Sex and evolution. Princeton Univ. Press, 1994. Evolution, weighting and phylogenetic utility of mito- Princeton, NJ. chondrial gene sequences and a compilation of conserved poly- Corresponding Editor: M. BjoÈrklund 1520 TOMI KUMPULAINEN ET AL. E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ 31 07 50 45 41 56 12 55 59 57 40 43 48 52 07 00 24 12 47 41 40 54 10 08 40 05 24 38 28 37 39 40 10 04 44 43 22 36 07 44 Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј 44 45 42 42 42 42 42 41 41 41 42 39 39 54 25 37 37 42 29 29 29 29 44 42 39 40 39 38 44 35 41 33 25 28 30 30 41 41 00 59 Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ N, 25 N, 25 N, 25 N, 25 N, 25 N, 25 N, 25 N, 25 N, 25 N, 25 N, 25 N, 25 N, 25 N, 25 N, 25 N, 25 N, 25 N, 25 N, 25 N, 25 N, 25 N, 25 N, 25 N, 25 N, 25 N, 25 N, 25 N, 25 N, 25 N, 25 N, 25 N, 25 N, 27 N, 27 N, 27 N, 27 N, 25 N, 25 N, 26 N, 25 Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ location Geographical 25 56 45 40 43 53 30 31 33 23 28 01 49 46 49 03 57 37 44 51 04 17 41 37 39 44 35 08 07 53 53 58 18 25 41 42 51 54 19 09 Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј 12 11 16 16 16 16 15 15 15 15 15 15 14 13 19 13 13 13 12 12 13 13 12 15 06 06 48 49 55 49 54 51 42 38 41 41 12 12 52 52 Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ 62 62 62 63 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 60 60 60 60 60 63 63 63 62 62 61 61 60 62 6 7 6 7 5 8 7 9 7 40 28 14 16 16 14 10 64 16 46 31 20 27 23 188 46 67 14 36 30 56 48 50 14 26 15 53 size 480 936 248 291 374 108 136 457 122 941 164 107 117 107 177 129 Sample 8 7 80 37 26 56 34 117 124 416 115 236 556 354 311 1999 2000 2001 7 8 5 5 7 7 7 10 15 15 15 20 10 14 11 10 10 21 15 20 20 20 10 15 190 90 30 30 27 13 80 30 30 70 30 40 26 28 40 31 30 65 74 30 40 11 40 30 40 30 30 27 25 of traps Number PPENDIX A 70 10 21 12 62 60 59 60 83 29 20 Ð 110 100 138 1999 2000 2001 ) ) ) ) ) ) ) ) ) ) )40 ) ) ) ) ) ) ) ) ) ) ) Picea Populus Picea Pinus Pinus Pinus Alnus Betula ( ( ( ( ( ( ( ) ) ( ) ) type Forest Pinus, Picea Pinus, Picea Pinus, Picea Pinus Sorbus, Picea Pinus, Betula Betula, Pinus Pinus Pinus, Betula Betula, Picea Pinus, Betula Pinus, Betula Pinus Sorbus, Betula Betula, Pinus Betula, Pinus Betula Populus, Betula ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( Picea, Pinus Picea Picea Picea Picea, Betula Picea Betula, Pinus Picea, Betula Betula Picea Betula Betula, Populus, Pinus Betula, Pinus, Alnus Betula, Picea Pinus Betula Betula Picea, Pinus, Betula Picea Picea, Betula Betula Picea Picea, Pinus Pinus, Betula Picea, Betula, Populus Picea, Pinus, Betula Picea Picea, Pinus Picea Picea, Betula Picea, Betula, Pinus Picea Picea Picea Picea Picea Picea, Pinus, Betula Betula, Picea Picea Betula, Pinus 60 m 35 m 100 m 25 m 15 m 30 m 40 m 35 m 40 m 40 m 100 m 40 m 150 m 35 m 25 m 25 m 35 m 20 m 20 m 40 m 40 m 70 m 70 m 30 m 30 m 40 m 25 m 25 m 40 m 50 m 30 m 30 m 25 m 30 m 40 m 30 m 45 m 50 m 40 m 100 m size ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ ϫ Habitat 5 25 25 30 30 25 50 30 15 45 20 10 25 35 30 15 20 25 25 20 20 20 25 30 15 20 25 25 15 15 20 30 10 15 20 20 20 30 25 10 Description of sampling and study locations. In forest type, main genera of trees are listed ®rst; genera in parentheses are less numerous. Èv Èhde Site Èja name Èr Ènk 2 Ènk 1 Yl. Kotaj Hanhij Ko Kanav Peta Keur 1 Kuusim 1 Kuusim 2 Kuusim 3 Kuusim 4 Sip 3 Laajav 6 Muur 1 Muur 2 Salama Tukki Villa Viljan Pennala Kaup Puhd Pyo Piha 1 Piha 2 Must 1 Must 2 Leivo 3 Leivo 4 Sip 1 Sip 2 Huvin Isa Psa Pihta 1 Laajav 1 Laajav 2 Laajav 3 Laajav 4 Laajav 5 Ko Site 1 2 3 4 5 6 7 8 9 number 13 14 15 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 41 42 47 48 10 11 12