* Department of Molecular Biology/Genetics, Ume University, Sweden
Department of Entomology, Institute of Zoology, Jagiellonian University, Krakow, Poland
Correspondence: E-mail: per.stenberg{at}molbiol.umu.se.
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Abstract |
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Key Words: clones polyploidy weevil Otiorhynchus scaber parthenogenesis
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Introduction |
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The traditionally advocated advantages of asexual reproduction are primarily a twofold increase in reproductive output and an increased ability for dispersal, since a single female suffices to establish a new population. There are, of course, also major disadvantages. On a short timescale, the inability of clones to create significant variation between generations is thought to render them poor competitors in a changing environment, as well as sensitive to parasites and diseases (e.g., Dybdahl and Lively 1998). In the long run, the lack of recombination should lead to an accumulation of deleterious mutations (Felsenstein 1974; Lokki 1976).
In parthenogenesis, the egg develops into a new individual without fertilization. Animals with both sexual and parthenogenetic forms often exhibit geographical parthenogenesis (Vandel 1928, 1940), so that sexual forms exist in a limited area, whereas outside of this area, only clones are found. However, in some species, with clonal forms, higher ploidy level is correlated to a wider distribution in the marginal areas, and sexual as well as all asexual forms coexist in a limited central area (Suomalainen 1940). Since this latter form of distribution is somewhat distinct from Vandel's definition, we will here use the term "geographical polyploidy" instead.
Otiorhynchus scaber
Among insects, weevils are well known to have a large number of parthenogenetic lineages (Suomalainen 1940; Suomalainen, Saura, and Lokki 1987). Parthenogenetic weevils are apomictic (i.e., they lack meiosis and recombination). Apomixis is the most common form of parthenogenesis. It has been observed in a number of major insect groups (i.e., Orthoptera, Diptera, and Coleoptera) (Suomalainen, Saura, and Lokki 1987). A particularly high frequency of apomictic lineages has been observed in the genus Otiorhynchus (Suomalainen 1940; Mikulska 1960) that has been studied in Central Europe since the 1920s (Penecke 1922; Szekessy 1937; Jahn 1941). Jahn (1941) observed that males in weevils with clonal forms only exist in areas of Europe not glaciated during the latest Ice Age, whereas all female populations dominated the surrounding areas. Many weevil species of the tribe Entimini have been studied using cytology since the 1930s and enzyme electrophoresis since the 1960s (Suomalainen, Saura, and Lokki 1987). We here use a molecular approach to investigate evolution in a clonal complex using the weevil Otiorhynchus scaber (Linnaeus, 1758).
O. scaber is a small (approximately 5 mm.) flightless weevil that lives most of the year below ground. It is considered a minor forest pest and feeds mainly on spruce and blueberry roots, as well as on young shoots of spruce (Stenberg et al. 1997). O. scaber has three traditionally recognized forms, which show a clear-cut geographic polyploidy (fig. 1). Diploid sexual individuals are only found in some parts of the Alps, whereas triploid clones inhabit most of the central European montane and submontane areas. Tetraploid clones have conquered almost every Palearctic spruce forest (fig. 1). Males have been observed to copulate with females of all forms, and the genitalia of sexual and parthenogenetic females do not differ (Szekessy 1937). Clonal diversity is highest in the central area of distribution and decreases rapidly toward the margins (Stenberg et al. 2000).
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Until now, it has been difficult to differentiate between these hypotheses because of the limited resolution of available data in earlier studies. We here aim to answer the following important and unresolved questions in the hope of illuminating the evolution in parthenogenetic weevils. (1) Has parthenogenesis arisen more than once? (2) Does clonality precede polyploidy or the other way around? (3) How are the different forms related to each other? (4) To what extent does the current distributions reflect phylogenetic relationships?
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Material and Methods |
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DNA Extraction, PCR, and Sequencing
DNA was prepared using Wizard genomic DNA purification kit (Promega). PCR was performed using Ready-to-Go PCR beads (Amersham Pharmacia Biotech Inc.). Three partial mitochondrial gene sequences were amplified: COI, COIII, and CytB (see Supplementary Material for primer information). The following PCR conditions were used: 15 cycles of 95°C (30 s), 60°C (30 s, 0.5°C decrease/cycle), and 72°C (1 min); 20 cycles of 95°C (30 s), 53°C (30 s), 72°C (1 min); and one cycle of 72°C (3 min). Amplified markers where sequenced with DYEnamic ET terminator kit (Amersham Pharmacia Biotech Inc.) and an ABI Prism 377 apparatus (PerkinElmer). To verify the quality of sequences, COI and COIII where reamplified and resequenced in 20 specimen. CytB where reamplified and resequenced in all individuals at least once. All electropherograms where manually examined and ambiguous sites assigned a question mark.
Alignment and Phylogenetic Analysis
Sequences were processed and aligned using Bioedit version 5.0.9 (Hall 1999) and ClustalW version 1.4 (Thompson, Higgins, and Gibson 1994) software. In two of the genes surveyed, COI and COIII, a continuous part of the genes was used for phylogenetic analysis (552 bp and 297 bp, respectively). After aligning the sequences, we removed character sites that had four or more sequence ambiguities. No character sites where removed from COI and COIII, from CytB 129 out of the 283 aligned base pairs where removed. The complete alignment contained no gaps. A total of 95 informative characters from all three genes resolve the ingroup, 21 from COI, 51 from COIII, and 23 from CytB.
Heuristic maximum-parsimony analysis with a total-evidence approach was performed using NONA version 2.0 (Goloboff 1998) in the Winclada version 0.99m24 software (Nixon 1999). Parameters in the heuristic search were 30,000 maximum trees, 30 replications with 20 starting trees per replication and multiple TBR + TBR (Tree Bisection and Reconnection). Branch length was examined on a subset of the most parsimonious cladograms using acctran (fast) optimization. Since we are mainly interested in tracing major mtDNA lineages, we summarized all most parsimonious cladograms in a strict consensus after collapsing unsupported nodes and deleting all suboptimal trees. This allows us to keep only unambiguously supported relationships. The topology of the consensus tree was then used for assessing the lowest possible number of origins of parthenogenesis (parsimony-based optimization, equivalent to Camin-Sokal optimization). Since we wanted to evaluate the heuristic search in the parsimony analysis and believe that it is favorable to use different approaches to examine phylogenetic information, a Bayesian analysis using the program MrBayes 2.01 (Huelsenbeck and Ronquist 2001) was also performed. MrBayes was run for 6,000,000 generations with a tree sample frequency of 1,000, four chains, gamma distributed substitution rate with six substitution parameters (general time reversible model), and variable base frequency parameters. The log-likelihood sum reached a stable value after approximately 1,000 trees. Out of the 6,000 trees acquired, a majority rule consensus was created from the last 4,000 trees in MrBayes.
Enzyme Electrophoresis
Since the distribution of diploids in the consensus tree greatly surprised us, we also examined 38 diploid females from the Mozirje and Plesch populations with enzyme electrophoresis. The specimens from Mozirje were collected at two separate occasions and were therefore analyzed separately. Four to six loci (see Supplementary Material) were screened according to Shaw and Prasad (1970) with minor modifications. Eleven percent starch gels were run as described in Westerbergh and Saura (1992). Fstat software version 2.9.3.2 (Goudet 2002) was used to calculate allele frequencies (see Supplementary Material). The likelihood (Psex) of sampling the multilocus genotypes (MLGs) found in the three samples, at least as many times as observed, in a panmictic population given the sample size was calculated using the MLGsim software (Stenberg, Lundmark, and Saura 2003). To obtain critical values for the test statistic, Psex, we increased the number of simulations in MLGsim until stable critical values were reached.
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Results |
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Parsimony analysis using one of the closest known relatives of O. scaber as outgroup (O. nodosus) resulted in cladograms with surprisingly unequal distribution of evolutionary changes (data not shown). Therefore, we sequenced three weevil species more distantly related to O. scaber (Strophosoma melanogrammum, O. lepidopterus, and O. singularis). An initial cladistic analysis with the most distant relative (S. melanogrammum) as outgroup was performed to determine the most appropriate outgroup species for the ingroup analysis. Subsequent parsimony analysis, using the most suitable outgroup (O. singularis), resulted in 5,452 equally parsimonious cladograms with a length of 166, an ensemble consistency index (Ci) of 68, and an ensemble retention index (Ri) of 96 (data not shown). Only five out of the 95 informative characters resolving the ingroup were nonsynonymous substitutions. To create the strict consensus, 10 branches were collapsed (fig. 2). Rooting with the other two species (S. melanogrammum and O. lepidopterus) resulted in the same ingroup topology in the consensus. The Bayesian approach resulted in a consensus that supports all but one of the major branches of the cladistic consensus with posterior probabilities above 90. The exception is the branch defined as mtDNA lineage B, which has a posterior probability of 58 (fig. 2).
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Statistical analysis of diploid females from Plesch and the two Mozirje samples clearly shows the presence of MLGs that are highly unlikely to be the result of sexual reproduction (table 1), indicating clonal origin.
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Discussion |
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Origin of Asexuality and Polyploidy
Only considering clades represented in the cladistic consensus, we find that asexual reproduction has originated at least three times in O. scaber (fig. 2). This may of course be, and probably also is, an underestimation since it only is possible to safely assess the lowest number of times asexuality must have originated. However, synapomorphies observed in pure parthenogenetic clades, lacking close sexual relatives (e.g., Mozirje clones [fig. 2]) are almost certainly derived from a sexual ancestor. It is more probable that the sexual ancestors of these groups have become extinct or simply are not sampled in our study, than a line of ancestor clones has accumulated the mutations. Considering this, we find it more probable that clonality has originated several times in these weevils. Interpretation of the number of times polyploidy has evolved depends on the conclusions concerning the origin of asexuality. Triploid forms of O. scaber are evidently polyphyletic and must have evolved at least three times, but the real number given our data is more likely to be at least five to six. Tetraploids are only found in lineage B and have originated at least one to three times, considering the unresolved relationships in the consensus (fig. 2).
Based upon the mtDNA analysis, we cannot deduce which path of evolution gave rise to the different ploidy levels. There is, however, demographic support for the hypothesis that diploid clones are directly derived from sexual populations. The fact that diploid clones are only found in areas where sexuals reside and that there are many Slovenian sexual populations where diploid but not triploid clones are found make it unlikely that the diploid clones are a derived form of asexuals. These data do not, however, rule out the possibility of an origin of polyploid asexuals directly from sexuals.
There are two different scenarios that may explain the apparent disjunct clustering of sexuals and clones collected from the same locality. The two scenarios are, however, not necessarily mutually exclusive. Lineage sorting and genetic isolation during glaciations has probably caused the haplotype diversity observed within and between lineage A and lineage B. In the interglacial periods, recolonization of previously glaciated areas may have resulted in an overlapping distribution of clones originating from different populations. Subsequent lineage sorting could then explain the lack of closely related clones in the Plesch and Mozirje populations. Alternatively, secondary contact between sexuals from lineage A and lineage B resulted in hybridization events, creating clonal forms retaining only the maternal haplotype.
The two Austrian areas where diploid sexuals are found, populations A13 and Plesch, are examples of well-documented forest refugia during the latest (Würm = Wisconsin) glaciations (Jahn 1941; Huntley 1990; Hewitt 1999). Between the Austrian refugial areas and Slovenia (which was not glaciated during the latest cold period) are high mountains that represent dispersal barriers. O. scaber populations are not found on higher ground on these mountains, even though there is spruce present. Our observations agree with the data compiled by Hewitt (1999) on the effect of the Pleistocene glaciations on the European biota. He found the Alps to be a dispersal barrier during cold periods and a major hybridization zone in the warmer periods.
Although mitochondrial markers are insufficient in discriminating between the two scenarios, there are other observations that can be interpreted to support hybridization within the complex. O. nodosus clusters within the ingroup, and it should be noted that the specimens are polyploid clones. Suomalainen (1940) and Seiler (1947) have shown that many parthenogenetic forms of weevils have separate chromosome sets that form separate metaphase plates during meiosis. In triploids, three separate haploid chromosome sets have been observed, as well as one diploid and one haploid set. These observations, together with the incredibly high species diversity of weevils with unusually large number of parthenogenetic forms, make it plausible that hybridization events are important in weevil evolution. In other words, one needs to be very careful when doing species phylogenies on weevils, especially if mtDNA is used.
Geographic Distribution and Phylogenetic Relationships
We do not want to make any precise deductions of the age differences between clonal lineages based upon our data. As mentioned earlier, parsimony optimization of asexuality may only ascertain a lowest number of origins in complexes where asexuality has originated several times. This in turn may affect the certainty of distances to the latest sexual ancestor of different clones, invalidating age approximations (Butlin 2002). It is, however, safe to conclude that the time to acquire the amount of sequence divergence present between the "A" and "B" lineages vastly surpasses the time since the latest Ice Age. This conclusion is based on the estimation of Ribera, Hernando, and Aguilera (2001) that a mean divergence rate in a beetle mtDNA of approximately 2% per million years. Consequently, the present distribution of O. scaber does not accurately reflect the phylogenetic relationships within the species. This can be clearly seen in populations (e.g., Plesch) where apparently unrelated clonal lineages coexist. An interesting observation in our data is that many clones clustering in basal parts of our tree have accumulated very few substitutions in the three mitochondrial regions studied here. We have interpreted this as a sign of lower mutation rate in clones, although we have no explanation of why it should be so.
Polyploid Success (the More the Merrier)
Polyploidy and gametophytic apomixis are always associated with each other in plants (Asker and Jerling 1992, p. 109). Botanists have, accordingly, no way of separating the two. Even though there are fewer cases in the Animal Kingdom, the study of animal complexes with clones of different ploidy levels allow for separating the effects of asexuality from the ones of related to polyploidy.
Ever since the 1910s, when Penecke (1922) started studying this weevil complex, diploid females or males have not been observed outside the areas where sexuals now reside. Our results and the discovery of diploid clones indicate that the reason for geographic polyploidy in O. scaber is mainly a consequence of polyploidy and not of asexuality in itself. The evident advantage of the polyploids cannot be attributed to the usual ecological or demographic positive effects traditionally attributed to asexuality (see, e.g., Law and Crespi 2002; Maynard Smith 1978) since the diploid clones should share the same advantages of asexuality. This pattern, also observed in other weevils and other animals, where higher ploidy level is correlated to wider distribution (Suomalainen, Saura, and Lokki 1987), supports the argument that ploidy level in itself is an important factor in the evolution of many parthenogenetic forms. We propose that evolutionary studies of clonal forms should not only concentrate on aspects of sexual versus asexual reproduction. We believe that adaptive effects of polyploidization in animals deserve far more attention.
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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