The significance of relatedness and gene flow on population genetic structure in the subsocial spiderEresus cinnaberinus(Araneae: Eresidae)

Biological Journal of the Linnean Society (1998), 63: 81–98. With 3 figures

The significance of relatedness and gene flow on population genetic structure in the subsocial spider Eresus cinnaberinus (Araneae: Eresidae) JES JOHANNESEN1, THOMAS BAUMANN2, ALFRED SEITZ1 AND MICHAEL VEITH1 1

Institut fu¨r Zoologie, Universita¨t Mainz, Saarstraße 21, D-55099 Mainz, Germany; Insitut fu¨r Zoologie, Martin Luther-Universita¨t Halle-Wittenberg, Kro¨llwitzer Str. 44, D-06099 Halle, Germany 2

Received 29 May 1997; accepted for publication 4 September 1997

Interdemic selection, inbreeding and highly structured populations have been invoked to explain the evolution of cooperative social behaviour in the otherwise solitary and cannibalistic spiders. The family Eresidae consists of species ranging from solitary and intermediate subsocial to species exhibiting fully cooperative social behaviour. In this study we, in a hierarchical analysis, investigated relatedness of putative family clusters, inbreeding and population genetic structure of the subsocial spider Eresus cinnaberinus. Five hierarchical levels of investigation ranging from large scale genetic structure (distances of 250 and 50 km level 1 and 2) over microgeographic structure (20 km2 and 4 km2, level 3 and 4) to a single hill transect of 200 m (level 5) were performed. The purpose of level 5 was two-fold: (1) to investigate the relatedness of putative family groups, and (2) to evaluate the influence of both family living and sampling design on higher level estimates. Relatedness estimates of putative family groups showed an average relatedness of R=0.26. There was no indication of inbreeding. In contrast to social spiders, genetic variation was abundant, He≈0.10. The population genetic structure was intermediate between social and asocial spiders. Genetic variance increased continually across hierarchical levels. Family structured neighbourhoods biased differentiation estimates among level 5 samples (FST=0.04) and level 3 and 4 samples (0.070, was caused by disjunct sampling from separate neighbourhoods. Larger scale samples were highly differentiated 0.12
ADDITIONAL KEY WORDS:—Arachnida – dispersal – isolation – evolution of sociality – allozymes.

Correspondence to: J. Johannesen. E-mail [email protected]. 0024–4066/98/010081+18 $25.00/0/bj970186

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 1998 The Linnean Society of London

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J. JOHANNESEN ET AL. CONTENTS

Inroduction . . . . . . . . . . Material and methods . . . . . . Sampling design . . . . . . . Electrophoresis . . . . . . . Genetic data analysis . . . . . Results . . . . . . . . . . . Genetic differentiation . . . . . Relatedness . . . . . . . . Discussion . . . . . . . . . . Relatedness and genetic structure . Implications for evolution of sociality Acknowledgements . . . . . . . References . . . . . . . . . .

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82 84 84 87 88 89 89 91 93 93 95 96 96

INTRODUCTION

Three main hypotheses have been put forward to explain altruistic behaviour: kin selection, group selection and reciprocity (Michod, 1993). Family structured kin selection models assume group-living of related individuals among which interactions take place (e.g. Wade & Breden, 1981; Michod, 1982; Uyenoyama, 1984). Such models have been a powerful tool to explain the evolution of social behaviour in the haploid-diploid genetic system of Hymenoptera (Michod, 1993). Individual selection, which within groups opposes the evolution of altruism, may be overcome by genetically homogenizing group members through inbreeding or interdemic selection and thereby increasing genetic variance among groups. Among diploid organisms group selection and/or inbreeding are thought to play an important role in the evolution of social behaviour leading to highly structured populations in, for example, termites (Reilly, 1987; Kaib et al., 1996), naked mole-rats (Reeve et al., 1990), shrimps (Duffy, 1996), and spiders (Lubin & Crozier, 1985; Smith, 1986; Roeloffs & Riechert, 1988; Smith & Engel, 1994; Smith & Hagen, 1996). In the study of evolution of social behaviour subsociality provides an opportunity to compare the genetic status of species which are intermediate between asocial and fully social. In contrast to ants and termites, which all are social, spiders show varying degrees of social behaviour. The majority of spiders are solitary, territorial and cannibalistic. In at least seven families a few species exhibit social cooperative behaviour (Avile´s, 1997), although within each family most species retain the original solitary and cannibalistic way of life. It therefore seems likely that sociality in spiders has arisen independently several times (Kullman, 1972; Wickler & Seibt, 1993). Social spiders show no morphologically different or sterile castes and most individuals within colonies reproduce (Riechert & Roeloffs, 1993). Social spiders therefore are not eusocial as are ants and termites. However, social spiders do experience conditions hypothesized as being prerequisite for eusociality among diploid organisms, namely gradual metamorphosis, extensive parental care (subsociality), and long lasting niches (Alexander, Noonan & Crespi, 1991). Kullmann (1972) defined three criteria with which to recognize a spider as social: tolerance, interaction and cooperation. The threshold step seems to be tolerance among reproductives (Kullmann, 1972). Evolution of social and altruistic behaviour in spiders has been explained in a variety of contexts. Individual selection and mutualistic advantage of snare-building

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and predation have been advocated for evolution of altruistic behaviour (Brach, 1977). However, cooperative hunting which is common to all social spiders is also thought just to be a consequence of an aggregated lifestyle, and not the driving force (Packer & Ruttan, 1988; Seibt & Wickler, 1988). Within the family Eresidae pedogenesis, i.e. retention of juvenile characters, has been put forward to explain tolerance among adults (Wickler & Seibt, 1993). This hypothesis, which is based on fewer moults by social than by subsocial Eresids to reach sexual maturity, may, on the other hand, be a consequence of reduced growth and not a precondition for sociality. Under good food conditions subsocial Stegodyphus lineatus will tolerate each other beyond the juvenile stage (Schneider, 1995). Interdemic selection, which involves differential colony extinction and proliferation relative to the individual, and inbreeding have been favoured in recent years as explanations of social evolution (Avile´s, 1986; Roeloffs & Riechert, 1988; Smith & Hagen, 1996). A phenomenon observed in all social spiders is a female biased sex ratio (e.g. Vollrath, 1982; Elgar & Godfray, 1987; Avile´s, 1986, 1993; Lubin, 1991). All social spider colonies propagate by budding or swarming of nest related females (Vollrath, 1982; Lubin & Robinson, 1982) and inbreeding is thought to be pervasive. High levels of inbreeding and relatedness among females would, in the light of local mate competition, bias the sex ratio towards the dispersing sex (Bulmer, 1986). Riechert and Roeloffs (1993) mention little or no courtship in social spiders which is expected without mate competition, and would be suggestive of inbreeding as there is no selective need for males to compete as all are genetically identical. However, mate competition is observed in several social spiders (Henschel, Lubin & Schneider, 1995). Genetic studies have found very little genetic variation within colonies. Indeed, neighbouring colonies may by fixed for alternative alleles (Lubin & Crozier, 1985; Smith, 1986; Roeloffs & Riechert, 1988; Smith & Engel, 1994; Smith & Hagen, 1996). The gap between cannibalistic and social spiders seems difficult to bridge. A few species of spiders are considered subsocial: they experience extended parental care and may, after dispersing from the natal nest, live communally for a period of their life. For these species no genetic research has been done and several questions arise in a study of intermediate sociality comparative to social species. Do intermediate social species outbreed or is there indication of inbreeding, are individuals distributed non-randomly, e.g. in family groups, or do they disperse widely? Eresus cinnaberinus (Oliv., 1789) (formerly E. niger Pet.) is a periodically social, or subsocial, spider. It belongs to the cribellate spider family Eresidae in which permanent social behaviour has been described from five species (Riechert & Roeloffs, 1993). Eresus cinnaberinus is found in a variety of dry biotypes, such as grassand heathland and stony steppe habitat (Baumann, 1996 and references therein). The subsocial phase of E. cinnaberinus is characterized by the first weeks the newly emerged spiderlings spend in the maternal nest. The female catches prey and feeds the young by regurgitation. When the female dies she is consumed by the spiderlings. Only after this extended tolerance phase do spiderlings disperse, most probably only within the close vicinity of the maternal nest (Ratschker, 1995). From now on they live solitary underground in silk-lined burrows. Cooperative predation and occasional cohabitation among juveniles has been reported (Holl & Reinbach, 1991). The tube webs are often found in a clumped distribution. It seems that once a burrow has been established an individual does not disperse any further. Upon reaching sexual maturity males disperse whereas females probably stay in their burrows. Baumann

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(in press), in a three year study of dispersal, collected only six females and juveniles in pitfall traps. In a mark-recapture study of dispersing males a median dispersal distance of 13 metres, with a maximum distance of 59.43 metres, was observed, N=540 (Baumann, in press). Ballooning has never been observed (Ratschker, 1995). In the study area E. cinnaberinus females live for about 4 years and males 2–3 years. In this study we investigated relatedness and gene flow within and among groups of E. cinnaberinus. The aim of this study was to estimate the amount and distribution of genetic variation, for comparison with that of asocial and social spiders in an attempt to infer possible genetic predispositions for a transition to more permanent social behaviour. More specifically we were interested in how to assess where a population, or rather deme, begins and ends, whether inbreeding take place, whether web clusters consist of related individuals and the extent of local mating. We applied two approaches. First we estimated relatedness of individuals within putative family groups and differentiation among these in a single habitat patch. Secondly, we compared more geographically widespread samples in order to determine larger scale differentiation. By employing these two approaches we wished to gain insight into how family structure and/or sampling artefacts may influence the results of the larger scale analyses, and thereby infer at which hierarchical level a population and/or deme can be defined. A family structure of living may bias the estimated differentiation among populations towards an apparently stronger degree of isolation than is actually present. If this is the case it would be appropriate to investigate the relatedness of individuals within groups and not just the differentiation among groups (Pamilo, 1984).

MATERIAL AND METHODS

Sampling design Samples were collected in three German states: Rheinland-Pfalz, Sachsen-Anhalt, and Thu¨ringen (Fig. 1 and Table 1). The sampling design consisted of five geographical levels of analysis (Fig. 2). Samples of levels 1–4, which explore larger scale differentiation, are designated with capital letters (RP and SA-A to SA-H) (Table 1), whereas samples of level 5, which investigates genetic structure along a 200 m hill transect, are designated with lower case letters (a–c). Samples were divided into sub-samples. Abbreviations of sub-samples of levels 1–4 were given an area coding RP or SA and a number, e.g. RP 1.1 or SA 4.1, where RP 1.1 signifies Rheinland-Pfalz sub-sample no 1 (∗.1), which is only included in level 1 analyses (1.∗), and SA 4.1 signifies sub-sample no 1, which is included in all analyses to level 4 (table 1). Level 5 sub-samples were numbered 5.1 to 5.15 (Fig. 3). Individuals of level 5 were only included in level 5 analyses. The first level analysis included all sampling locations. In Rheinland-Pfalz collections were made from six locations. These consisted of three central localities (RP-1 to 3) with a distance of c. 4 km and three outlying localities c. 50 km away. These six locations were considered sub-samples of just one Rheinland-Pfalz sample since Rheinland-Pfalz in southwest Germany was used only as an outgroup comparison for large scale differentiation. The second level included the sub-samples

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Figure 1. A schematic presentation of geographic locations, with a map of level 3 (Halle) and, in the upper left hand corner, level 4 (Gimritz) sampling locations. Localities were situated in RheinlandPfalz (RP) and Sachsen-Anhalt/Thu¨ringen (SA and TH). Ellipsoids (SA-D to SA-H) indicate samples with 2–4 sub-samples, circles (SA-C) with one sub-sample.

from Kyffha¨user in Thu¨ringen, and Glu¨cksburger Heide and Halle in SachsenAnhalt. The greatest distance between level 2 samples was similar to the RheinlandPfalz samples, about 50 km. The third and fourth level of analysis was performed at Halle (Fig. 1). This area consists of porphyry and calcareous hills disjunctly distributed in the landscape. Hills are separated by farm land or urban development. The third level constisted of samples from Franzigmark (SA-D), the sample SA-C, and Gimritz (SA-E to SA-H). The Gimritz Nature Reserve (level 4) consisted of four samples collected along hill slopes. Samples from Gimritz consisted of 2–4 subsamples of 8–10 individuals. A total of 14 sub-samples were obtained. A sub-sample was collected within an area of approximately 10×10 metres. Caution was taken not to collect from adjacent webs. The distance between sub-samples within a sample was 100–150 m. The habitat between sub-samples was not in all cases continuos, i.e. apparent sub-optimal habitat could divide these. The pattern of subsampling at Gimritz was repeated for the slope at Franzigmark (three sub-samples), 4 km distance. Additionally, two sub-samples were obtained from the isolated patches SA-3.1 and SA-3.2 about 1 and 4 km from Franzigmark, respectively. In the data analysis these two sub-samples were treated as one sample, SA-C (Table 1) to enhance sample size.

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T 1. Sample locations and sample sizes. Abbreviations include an area coding RP or SA and a number, e.g. RP 1.1 or SA 4.1, where the first digit indicates the sampling level, and where the second digit is the sub-sample of a given level. See also Figure 1 for map locations Sample location R-P (RP) NSG Martinstein Kunoweg, Schoßbo¨ckelheim Idar-Oberstein NSG Rotenfels, Bad Mu¨nster am Stein NSG Pommern Battenberg, Pfalz S-A/T¨  (SA) Kyffha¨user Glu¨cksburger Heide Halle Lunzberg Brandberg Franzigmark Franzigmark 1 (Stahl) Franzigmark 2 (Draht) Franzigmark 3 (Beton) Gimritz Teichgrund Callunetum Teichgrund oben Teichgrund DFL Teichgrund Zufahrt Saalehang Zornberg Lauchengrund su¨dl. Eisenbahn Lauchengrund Kuppe I36 Su¨dspitze Kuppe I3 Wa¨ldchen Hang su¨dl. Wittsack Holl-Hang Hollunder-Hang 1

Abbreviation

Sample designation

Sample size

Sub-sample size

RP-1.1 RP-1.2 RP-1.3 RP-1.4 RP-1.5 RP-1.6

RP

60

14 7 11 11 11 6

SA-2.1 SA-2.2

SA-A SA-B

10 24

10 24

SA-3.1 SA-3.2

SA-C

18

8 10

SA-3.3 SA-3.4 SA-3.5

SA-D

30

10 10 10

SA-4.1 SA-4.2 SA-4.3 SA-4.4 SA-4.5 SA-4.6 SA-4.7 SA-4.8 SA-4.9 SA-4.10 SA-4.11 SA-4.12 SA-4.13 SA-4.14

SA-E

37

SA-F

40

SA-G

38

SA-H

20

8 9 10 9 10 10 10 10 10 10 10/1591 8 10 10

Individuals for level 5 analysis.

Finally, analysis at the fifth and lowest level took place at Wa¨ldchen in the Gimritz Nature Reserve along a 200 m hill transect. Here the Gimritz sampling design was repeated, with a similar number of sub-samples (15) and individuals (159) (Fig. 3). Each sub-sample (5.1–5.15) corresponded to a putative family group. Three samples were defined. A main sample, sample b, of nine sub-samples was situated in the centre of the transect. These sub-samples were collected within an area of approximately 5×40 metres. Samples a and c were situated 25 m and 80 m from sample b, respectively. Samples a and b were divided by apparently sub-optimal habitat. The area of sample a was 2×4 meters; of sample c 5×10 meters. Subsamples consisted of 5–22 individuals. For all individuals located coordinates and age were registered. In contrast to the situation at Gimritz, individuals from subsamples were often collected in the immediate vicinity of each other. The individuals of the fifth (Wa¨ldchen) level were not included in the higher (1–4) level analyses in order to avoid sample size biased results.

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Level 1. Total Rheinland-Pfalz (6)

250 km

Sachsen-Anhalt (20) .. Thuringen (1)

.. Level 2. SACHSEN-ANHALT/THURINGEN ..

GucksburgerHeide (1) .. Kyffshauser (1)

30–50 km

Halle (19)

2

Level 3. HALLE (20 km ) Franzigmark (3) Brandberg (1) Lunzberg (1)

c. 5 km

Gimritz (14)

2

Level 4. GIMRITZ (4 km ) Gimritz, 4 samples–14 samples 4 subsamples

4 subsamples

2 subsamples

4 subsamples

Distance between samples 0.5–2 km

.. Level 5. WALDCHEN (200 m transect) ..

Waldchen (SA-4.11) 3 samples–15 sub-samples* 3 sub-samples

25 m

9 sub-samples

80 m

2 sub-samples

*1 sub-sample not included in 3 sample FST estimation

Figure 2. Hierarchical sampling design for Eresus cinnaberinus consisting of five levels. The number of sub-samples within levels 1–3 are given in brackets. Location names are given in Table 1.

Electrophoresis Spiders were stored in liquid nitrogen. Abdominal tissue was homogenized in 80 ml Pgm-buffer (Harris & Hopkinson, 1978) by ultrasound and centrifuged at 14 000 rpm for 2 minutes. Analysis was done by cellulose acetate electrophoresis (Hebert & Beaton, 1993). A total of 18 enzyme systems representing 24 loci could be scored: Aat-1, -2 (EC 2.6.1.1), Acon (EC 4.2.1.3), Adh (EC 1.1.1.1), Ak (EC 2.7.4.3), Apk-1, -2 (EC 2.7.3.3), Fum (EC 4.2.1.2), G-3-Pdh (EC 1.2.1.12), G-6-Pdh (EC 1.1.1.49), Gpd (EC 1.1.1.8), Hbdh (EC 1.1.1.30), Hk (EC 2.7.1.1), Idh-1, -2 (EC 1.1.1.42), Ldh (EC 1.1.1.27–28), Mdh-1, -2 (EC 1.1.1.37), Mpi (EC 5.3.1.8), Pep-B 1,-2,-3 (leucine-glycine-glycine) (EC 3.4.11 or 13), Pgi (EC 5.3.1.9), Pgm (EC 5.4.2.2). Three buffer systems were used: Tris-Maleic acid pH=7.0 (adjusted from TM

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8

5.3

5.7 4

5.14

5.5 b

5.2 5.6

Metres

0 –4

5.15

c

5.9 5.8 5.1 5.4 5.12 5.11 –8

a

5.10

–12 5.13 –16 –40

0

40

80

120

160

Metres

Figure 3. Level 5 transect sample for relatedness estimates at Wa¨ldchen. The letters a, b, and c refer to samples for F-statistics. Numbers 1–15 refer to sub-samples for F-statistics and relatedness estimates. The four demes for relatedness estimates (Table 4) consisted of three samples a, b, and c and the isolated sub-sample 13.

pH=7.8, Colgan 1986) for Acon, Ak, Apk, G-6-Pdh, Gpd, Hbdh, Ldh, Pgi; Pgm; TrisCitrate pH=8.2 (Richardson, Baverstock & Adams, 1986) for Fum, G-3-pdh, Idh, Mdh; Tris-Glycine pH=8.5 (Hebert & Beaton, 1993) for Aat, Adh, Mpi, Hk, Pep-B. All enzymes were run for 30 minutes. Adh, Ak, Gpd, Hbdh, and Pep-B were run at 200V; all others at 250V. Genetic data analysis Putative loci were analysed only if they displayed patterns consistant with known quaternary structure (Richardson et al., 1986). If a locus only showed one band it was interpreted as monomorphic. The furthest migrating allele was designated 1, the second furthest 2, and so forth. Loci were tested for departure from expected Hardy-Weinberg proportions with the Louis–Dempster (1987) exact test. Exact probability genotypic linkage disequilibria based on the algorithim of Weir (1991) were tested for all subsets of samples. Allelic distribution patterns were analysed with the exact probability test of Raymond & Rousset (1995a). This test was only applied in the third and fourth level analyses for the comparison and interpretation of allele distribution patterns within and among hills. The above tests were all tested with the program GENEPOP (Raymond & Rousset, 1995b). Genetic differentiation among samples was analysed at different levels of regional subdivision with the estimates of FIS, FIT and FST by employing the F-statistics of Weir & Cockerham (1984). A hierarchical distribution of genetic variation was quantified with Wright’s (1978) hierarchical F-statistics. At level 5 (Wa¨ldchen), F-statistics were obtained both for the three samples and for the 15 sub-samples. Standard deviations were obtained using the jack-knife procedure. Finally, isolation by distance (Slatkin, 1993) was investigated among sub-samples of level 5 to evaluate sub-sample relationships.

GENETICS OF A SUBSOCIAL SPIDER

89

Under the equilibrium assumption of Nem=(1/FST-1)/4, the number of migrants per generation (Nem) between sample pairs can be calculated. A negative linear correlation between log (Nem) and the log of the geographical distance (i) indicates isolation by distance. Since pair-wise Nem estimates are not independent, the association between log (Nem) and log (i) was tested with a Mantel test (subroutine Mantel from the program package GENEPOP; Raymond & Rousset, 1995b). Relatedness estimates were obtained using the program Relatedness 4.2b (Queller & Goodknight, 1989). Estimates were tested for significance by a one-tailed t-test using the number of included sub-samples as degrees of freedom. Between age-class estimates were tested with two-tailed t-tests. All estimates were weighted by total individuals. All relatedness estimates were tested with and without respect to deme division. At level 5 (Wa¨ldchen) sub-samples were divided in to four demes. The four demes correspond to samples a–c of the FST estimates, and the isolated cluster 5.13 (Fig. 3). Average population, age-class, and nearest group relatedness were analysed. Relatedness estimates from level 3 and 4 (Halle and Gimritz) included samples and sub-samples from samples SA-D to SA-H.

RESULTS

Genetic differentiation Levels 1 and 2 Across all samples, 18 of the 24 loci investigated were polymorphic (allele frequency tables provided on request). For all sub-samples no significant deviations from Hardy-Weinberg proportions were observed. However, due to small sub-sample size and the conservative nature of Hardy-Weinberg tests and this finding might not be unexpected. The average sub-sample heterozygosity was significantly lower in Rheinland-Pfalz, He=0.060 (range: 0.007–0.106), than in Sachsen-Anhalt/Thu¨ringen, He=0.094 (range: 0.024–0.144), P<0.05. There was no indication of inbreeding, FIS≈0, when comparing sub-samples. However, among samples FIS was significantly greater than zero (Table 2). Genetic differentiation among sub-samples of the two German regions (level 1) was high, FST=0.26 (Table 2). This was primarily caused by fixation of the Gpd 2allele, an increased Pgm 1-allele frequency, and no Fum variation in Rheinland-Pfalz (Table 2). Repeating the analyses for the samples (we treated Rheinland-Pfalz as one sample) we found almost the same FST=0.22, indicating that level 1 analysis was truly influenced by an among region variance and not the division of samples into sub-samples. The hierarchical analysis of genetic variance (Wright, 1978), based on sub-samples, indicated about 60% of the variance estimate was caused by regional separation, and 40% within regions (Table 3). Differentiation among sub-samples within Rheinland-Pfalz, FST=0.17±0.04, was comparable to that of sub-samples within Sachsen-Anhalt, FST=0.18±0.03. Levels 3 and 4 Decreasing the geographical range from level 3 (20 km2) to level 4 (4 km2) also decreased the differentiation estimate, e.g. sample FST=0.12 to 0.07 (Table 2). The inbreeding estimate, FIS, was not significantly different from zero when estimated

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T 2. F-statistics for different sampling schemes and Hardy-Weinberg (HW) proportions for levels 3 to 5. HW estimates are based on all individuals samples within a level. N=number of sub-samples or samples N

FIT

FIS

FST

27 9

0.30±0.07 0.32±0.08

0.05±0.03 0.12±0.03

0.26±0.07 0.22±0.07

6 21

0.18±0.04 0.22±0.03

0.02±0.06 0.05±0.02

0.17±0.04 0.18±0.03

Level 3 Halle, sub-samples Halle, samples

19 6

0.21±0.04 0.21±0.04

0.01±0.04 0.11±0.03

0.20±0.04 0.12±0.03

Level 4 Gimritz, sub-samples Gimritz, samples

14 4

0.16±0.05 0.17±0.06

0.03±0.04 0.10±0.04

0.13±0.02 0.07±0.02

Level 5 Wa¨ldchen, sub-samples Wa¨ldchen, samples

15 3

0.02±0.02 0.02±0.03

−0.14±0.03 −0.02±0.02

0.13±0.03 0.04±0.02

Level 1 All sub-samples All samples Level 2 Rheinland-Pfalz Sachsen-Anhalt/ Thu¨ringen

HW

 v2=149, df 32, P<0.001  v2=60, df 28, P<0.001  v2=13, df 22, P=0.92

T 3. Hierarchical FST (Wright, 1978) for all sub-samples and for level 3 (Halle) samples (SA-C to SA-H). FSR signifies the differentiation among sub-samples or samples relative to a regional level. FRT is the differentiation within the regional level relative to the total level. The regions of all sub-samples were Rheinland-Pfalz, Glu¨cksburger Heide, Kyffsha¨user, and Halle. At Halle three regions based on sample locations were defined, (1) SA-C, (2) SA-D, (3) SA-E - SA-H FXY

VC

All sub-samples N=27 FSR FRT FST

0.180 0.111 0.271

0.481 0.333 0.813

Halle, samples N=6 FSR FRT FST

0.053 0.050 0.100

0.151 0.299 0.148

VC variance component.

among sub-samples, but was when estimated among samples. Hierarchical FST statistics for level 3 (Halle) showed 50% variance within and between areas (Table 3). In these tests we treated the two outlying sub-samples, SA 3.1 and SA 3.2, as one sample (SA-C). Hereby we gain a conservative differentiation estimate, because by increasing the genetic variation within a sample, the among sample differentiation estimate decreases. This sample was not in Hardy-Weinberg proportions, P<0.05. This is not unexpected as the two sub-samples are separated by a distance of 4 km. None of the remaining five samples (SA-C to SA-H) at Halle deviated significantly from expected Hardy-Weinberg proportions. In contrast, the allelic distribution

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among sub-samples within samples were significantly different from another. Deviations from expected Hardy-Weinberg proportions were found both for the total Halle sample and for the Gimritz sample (Table 2). Level 5 The Wa¨ldchen co-ordinate sample (159 individuals, 79 age-class 1, 57 age-class 2, 23 age-class 3+) did not differ significantly form Hardy-Weinberg proportions, P=0.92 (Table 2). Hardy-Weinberg proportions were also observed for both ageclass 1, P=0.99, and age-class 2 P=0.97. Of a total of 66 possible pairwise locus genetic linkage disequilibria comparisons thirteen were significant. The allele distribution was significantly different between age-class 1 and 2. Differentiation estimates among samples a–c (Fig. 3) were calculated in two ways: first including all individuals, second by only employing the first 30 individuals collected from sample 2, with the purpose of keeping the among sample size nearly equal. FST estimates were equal indicating slight structuring, FST=0.04. Neither FIS nor FIT were significantly different from zero. The allelic distributions among samples, for both calculations, were significantly different from one another. For the 15 sub-samples a high degree of structure, FST=0.13, and a significant excess of heterozygotes, FIS=−0.14 were observed (Table 2). The allelic distribution was highly significantly different among sub-samples. However, FIT=0 indicated that defining level 5 sub-samples as populations was erroneous and that individuals were part of just one hill population. This finding corroborated the Hardy-Weinberg proportions.

Relatedness Level 5 The observed differentiation among sub-samples observed of level 5 could in large part be explained by the sampling of related individuals within sub-samples. The average sub-sample relatedness estimate was R=0.287, which, in the absence of inbreeding (positive assortative mating), corresponds to individuals that on average were related as half-sibs (Table 4). There were no indications of inbreeding, F=0, thus the estimates were not caused by substructure or isolation by distance. Low sub-sample estimates of R were influenced by grouping different age-classes. The tentative division of level 5 into four demes was not justified. The four deme relatedness estimate did not differ from the one deme estimate (Table 4). This contrasts with the results from the level 3 relatedness investigation (see below). Due to too few age-class 3+ individuals we only analysed relatedness within this age-class and not relative to age-classes 1 and 2 and two-sub-sample comparisons. Two year olds, R=0.320, were significantly more related to one another than were one year olds, R=0.206, and three year olds, R=0.180, and the population estimate was therefore influenced quite strongly by the two year olds. A high variance was found within and among all age-classes; and especially so for age-class 1 (Table 5). In half of the sub-samples, R≈0; for the other half, R≈0.30–0.60, which is in the range of half to full-sibs. Relatedness between age-classes 1 and 2, R=0.114, (Table 4) was significantly less than among two year olds, P<0.001, and nearly significant, P=0.06 than among one year olds. Again, this nearly significant result was influenced

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T 4. Relatedness estimates, R±standard error taken over subsamples, N, included in the analysis, and inbreeding estimate F. All estimates are weighted by total number of group individuals. Estimates were gained for all sub-sample members (all), 1, 2 and 3 year olds, relatedness between 1 and 2 year olds (1,2 year) , and relatedness of subsample individuals including individuals of the nearest sub-sample (2sub). The Halle estimate omits sample SA-C. Maximum number of subsamples possible were for Halle 17, Gimritz 14, and Wa¨ldchen 15 Category

N

R

F

Level 3 (Halle) 5 demes 1 deme

17 17

0.163±0.044 0.243±0.033

0.095±0.034 0.145±0.022

Level 4 (Gimritz) 4 demes 1 deme

14 14

0.176±0.055 0.228±0.041

0.126±0.037 0.158±0.027

Level 5 (Wa¨ldchen) 4 demes all 4 demes all 2sub 1 deme all 1 deme all 2sub 1 deme 1 year 1 deme 1 year 2sub 1 deme 2 year 1 deme 2 year 2sub 1 deme 3 year 1 deme 1,2 year 1 deme 1,2 year 2sub

14 14 15 15 11 13 8 9 5 7 11

0.279±0.070 0.015±0.053 0.128±0.081 0.015±0.053 0.287±0.055 0.013±0.039 0.145±0.065 0.013±0.039 0.206±0.103 0.054±0.062 0.096±0.094 0.054±0.062 0.320±0.065 0.005±0.095 0.297±0.062 0.005±0.095 0.180±0.111 −0.098±0.110 0.114±0.078 0.025±0.040 0.009±0.069 0.006±0.040

T 5. Sub-sample relatedness estimates for different age-classes. N=total number of sub-sample individuals. N (∗) and R (∗) are the number of age-class individuals (1, 2, or 3) and their relatedness, respectively. (—) signifies a group with one or no individuals of that age-class and analysis not possible Sub-sample

N

N (1)

5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 5.10 5.11 5.12 5.13 5.14 5.15

22 8 10 5 11 13 10 5 15 7 13 10 10 10 10

6 2 2 5 10 13 2 — 15 — — 9 10 4 —

R (1) 0.13 −0.29 0.43 0.06 0.57 0.43 −0.44 — 0.30 — — 0.57 0.01 0.03 —

N (2) 9 — 7 — — — 8 4 — 6 8 — — 4 6

R (2) 0.41 — −0.01 — — — 0.30 0.08 — 0.68 0.46 — — 0.05 0.34

N (3)

R (3)

6 5 — — — — — — — — 5 — — 2 3

0.03 0.02 — — — — — — — — 0.44 — — 0.06 0.38

by the high age-class 1 variance. Including the nearest sub-sample (Table 4: 2sub) caused the relatedness estimate within age classes to decline significantly for ageclasses 1 and 1×2, P<0.01, but not for age-class 2 (Table 4). Isolation by distance tests mirrored relationships within sub-samples. All tests

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T 6. Isolation by distance regressions among level 5 (Wa¨ldchen) samples. A negative linear correlation between log (Nem) and the log of the geographical distance (i) indicates isolation by distance. Note: P values are based on Mantel tests, not regression analyses Sub-samples included All Sample b Samples a and b Samples b and c

Regression

P

logNem=0.478–0.147 log(i) logNem=0.912–0.692 log(i) logNem=0.600–0.384 log(i) logNem=0.673–0.202 log(i)

0.300 0.120 0.012 0.300

showed a negative correlation between gene flow and geographic distance. A significant correlation was observed only for sub-samples of samples a and b, P= 0.012. Within sample 1 a negative, but non-significant correlation was observed, P=0.12 Mantel test (Table 6). Across all sub-samples no isolation by distance was found. This finding was most likely caused by sub-samples of sample c, which were situated furthest away and showed low within sub-sample relatedness. Levels 3 and 4 The samples of Halle (omitting sample SA-C) and Gimritz were analysed with and without respect to deme (corresponding to a sample) structure. Without division into demes relatedness among sub-sample individuals seemed high, R=0.243 and R=0.228 (Table 4). However, the inbreeding estimate, F, was significantly greater than zero revealing that the relatedness coefficient was caused by population division and not by family members within sub-samples. Grouping sub-samples into demes (samples) caused the relatedness of sub-sample individuals to decrease, R=0.163 and R=0.176. However, F remained greater than zero, which indicated, as did the FST estimates of level 3 and 4, that sub-samples within samples were collected from separate neighbourhoods.

DISCUSSION

Relatedness and genetic structure Family clustering coupled to a secondary effect of subdivided habitat proved the differentiation of E. cinnaberinus samples intermediate between social, FST≈0.50–0.80 (Lubin & Crozier, 1985; Smith, 1986; Roeloffs & Riechert, 1988; Smith & Engel, 1994; Smith & Hagen, 1996) and asocial spiders FST≈0–0.15 (Galindo-Ramirez & Beckwitt, 1983; Riechert, 1993; Ramirez & Fandino, 1996; Ramirez & Froehlig, 1997, Johannesen and Veith, unpublished [most allozyme studies in asocial spiders deal with phylogenetic relationships and intraspecific differentiation are difficult or not possible to assess, e.g. Pennington, 1979; Cesaroni et al., 1981; Terranova & Roach, 1987; Rowell, 1990; Steiner & Greenstone, 1991; Colgan & Gray, 1992]). As in most outbreeding species there was no indication of inbreeding and levels of genetic variation were high compared to social spiders. The initial definition of a cluster of webs as a group of related individuals was only speculative. However, the age similarity indicated, and genetic analyses

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confirmed, that many web clusters consisted of related individuals. Level 5 analyses indicated family or sib-groups in three ways: (1) a significant sub-sample excess of heterozygotes FIS<0, compared to FIT=0; (2) genetic linkage disequilibrium at 20% of the pairwise locus comparisons caused by sampling sib-groups; and (3) the population estimate of R=0.26 showed a degree of relatedness. Different relatedness estimates within age-classes, two year olds R=0.30, one year olds R=0.21, and three year olds R=0.18, likely occurred because we defined the putative sub-samples as family groups, but can also be explained by differential dispersal of sibs between years. Spiderlings from neighbouring nests may disperse and settle in the vicinity of each other. This probably led to both a high within age-class variance of R and the differences among age-classes. The observed isolation by distance among sub-samples of level 5 indicated that females are sedentary and thus suggests a vertical female genealogy. Relatedness between age-classes, within samples and among neighbour groups increased both inter- and intrademic variance causing a microgeographic genetic structure. High differentiation among social spider clusters originate due to closed colonies (overview by Riechert & Roeloffs, 1993), whereas the E. cinnaberinus system is open. Within samples of levels 3 and 4 some of the sub-samples were rather discretely distributed and FIS>0 showed a Wahlund effect on a small geographic scale. This finding was likely due to disjunct sampling and contrasted the results of level 5 (Wa¨ldchen) where no indication of inbreeding was found, FIS=0 among samples a–c. Disjunct sampling of levels 3 and 4 sub-samples was confirmed in the level 5 analysis which showed that along a transect the individual heterozygosity relative to total heterozygosity was zero, FIT=0. As seen in the relatedness estimates, and an isolation by distance among sub-samples, the slight structuring of level 5, FST>0, was caused by a sampling artefact of family living (and probably limited female dispersal). As mentioned above, a Wahlund effect at Halle and Gimritz indicated inbreeding within samples SA-D to SA-H, but within sub-samples no inbreeding was observed. Thus, family clustering also biased the FST estimates of levels 3 and 4 where individuals within samples probably were collected from different neighbourhoods. Instead of family colonies as in social spiders one could speak of neighbourhoods of E. cinnaberinus. On the other hand, the Hardy-Weinberg disequilibrium of the total level 4 population (Gimritz) contrasted the results of the level 5 (Wa¨ldchen) population indicating at level 4 (4 km2) a true subdivision of samples. This subdivision likely originated as a consequence of limited dispersal and was enhanced by a patchy habitat. The subdivision was corroborated by Wright’s hierarchical analysis (Table 3). For an dispersing outbreeding organism within a small area as Gimritz (4 km2) and Halle (20 km2) differentiation was very high, and indicated little gene flow within a few kilometres. What then defines a population and/or a deme of E. cinnaberinus? This question can not be answered in a straightforward manner because of both a within patch and an among patch variance component. By increasing the sample size (relative to the sub-sample) and the geographical range the differentiation as expected declined (e.g. Gimritz sample versus Gimritz sub-sample) because more variation was included within each sample and because we may have put adjacent more related neighbourhoods in different samples. Thus, the among patch differentiation estimate is biased from the sedentary life mode of E. cinnaberinus. The differentiation at Gimritz must lie between the sample and sub-sample FST estimate, 0.08–0.18. As sub-samples likely often belonged to separate neighbourhoods the estimate probably lies in the

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upper half. Our tentative answer would be, despite the Wahlund effect, to characterise Gimritz (level 4) as a single population divided into patch demes, where a deme is defined as the level of random mating (FIT=0 and no ‘deme’ effect on R within the patch Wa¨ldchen). Each patch deme is neighbourhood structured, which is the reason for the high level of differentiation. This genetic structure could be extrapolated in a hierarchical fashion to all higher levels and differentiation expands as concentric rings from level 5 to level 1. It contrasts the findings for social spiders, where variation is related to colonies but not to geographical areas (Lubin & Crozier, 1985; Smith, 1986; Roeloffs & Riechert, 1988; Smith & Engel, 1994; Smith & Hagen, 1996). Only between levels 3 and 2 was no additional increase in variance observed. This was probably due to collecting only one sample within each of two areas. One reason could be that the great structuring within level 3 (Halle) will not cause much additional variance at only 30–50 km distance. The similar differentiation estimates within Rheinland-Pfalz and Sachsen-Anhalt/Thu¨ringen could support this explanation. However, because E. cinnaberinus is neighbourhood structured means that collecting only one sample per area may give a false estimate of an area’s genetic variation, and thus the variance among areas. In either case, due to the neighbourhood structure of E. cinnaberinus, the sampling of only two outlying samples (from different areas) for inclusion in level 2 analysis proved inadequate to evaluate differentiation at this level. Spiders collected from the Western and Eastern German regions (level 1) suggested little, if any, gene flow between these. Implications for evolution of sociality in spiders All social spiders experience female dispersal or founding of new colonies by propagules of parent colonies (Vollrath, 1982). In contrast, in asocial spiders it is the males that disperse and seek mates (Foelix, 1981). Males of the likewise subsocial spider Stegodyphus lineatus also disperse (Y. Lubin, pers. comm.). The mode of dispersal thus seems essential for social evolution in spiders. Shifting dispersal from males to females may increase the potential relatedness of offspring and inbreeding might cause cooperative behaviours to evolve. This implies inbreeding, high intergroup variance, and maybe evolution of social behaviour by interdemic selection (Roeloffs & Riechert, 1988; Smith & Hagen, 1996). Estimates of relatedness within colonies of Agelena consociata have been estimated to 0.556 (Riechert & Roeloffs, 1993) and an inbreeding coefficient of 0.69 in Stegodyphus dumicola has been reported (Wickler & Seibt, 1993). Evidence from E. cinnaberinus showed it to be an outbreeder, with high levels of genetic variation, and high intergroup variance (on the scale of an outbreeding organism). Within a deme relatedness estimates showed family clustering and restricted dispersal. We may be witnessing two structuring processes. First, increasing relatedness among neighbour females creating family neighbourhoods, and second outbreeding caused by dispersing males. It is conceivable that by extending the neotene period clusters of related spiderlings will sustain tolerance, but male dispersal persists and opposes intrademic relatedness over generations. For kin selection or inbreeding to propagate sociality the mode of dispersal must change too. In this sense a male–female conflict may, in terms of establishing an increased genetic variance among lineages, halt the evolution of social behaviour of E. cinnaberinus. For interdemic selection to mediate permanent social conditions by increasing

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relatedness through inbred groups turnover must be high, although a patchy habitat can compensate by increasing genetic variance (Slatkin, 1981). Interdemic selection for cooperative behaviour in spiders has been invoked primarily for social Agelenidae and Theridiidae (Lubin & Crozier, 1985; Smith, 1986; Riechert & Roeloffs, 1993; Smith & Hagen, 1996), whereas sociality in Eresids may be due to individual selection by pedogenesis (Kraus & Kraus, 1990; Wickler & Seibt, 1993), and may be driven by a different selection pressure. In an interdemic selection model for social behaviour, as has been suggested for Agelena consociata, aggression between males is scarce, which would be expected if males are highly related because no selective advantage among identical males exists (Riechert & Roeloffs, 1993). On the other hand, in social Eresids female choice or male-male competition is often observed (Henschel et al., 1995). Competition behaviour is observed in E. cinnaberinus too, and dispersing males have been observed to engage in fights (T. Baumann, unpublished). However, it is possible that one selection regime initiates an evolutionary process and another takes over once a certain limit is reached. For example, once tolerance has been achieved by individual selection in outbreeding populations, selection of family groups may take over. Intermediate sociality may be a distinct social condition and not an ancestral stage towards sociality. We do not know whether E. cinnaberinus is still evolving towards permanent sociality or has reached its social limits. Communal breeding in bees and wasps probably should not be viewed as a route to eusocial conditions (Packer, 1993). Lack of inbreeding or kin selection has been showed for several communal bees (Kukuk & Sage, 1994; Danforth, Neff & Barretto-Ko, 1996). Although the dispersal modes among social spiders and the intermediate social E. cinnaberinus differ, females versus males, it is likely female behaviour of both that cause high levels of differentiation among populations.

ACKNOWLEDGEMENTS

We thank Lorenz Becker for helping collecting spiders, Jes Søe Pedersen for helping with the Relatedness 4.2b estimates, and Yael Lubin and Jutta Schneider for discussions on the manuscript. Martin G. Ramirez and an anonymous reviewer made valuable comments on the manuscript. Permissions for collections were provided by Regierungsbezirk Koblenz and Regierungspra¨sidium Halle. The study was supported by the German Federal Ministry of Science and Technology (grant no. 339519A).

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