Mutations As Molecular Tools: the Metabolic-Rate Dependent Molecular Clock and DNA Barcoding of Allied Species

Mutations as molecular tools: The metabolic-rate dependent molecular clock and DNA barcoding of allied species

Lena Alena Brüstle

Zoological Museum Finnish Museum of Natural History University of Helsinki Finland

Department of Biological and Environmental Sciences Faculty of Biosciences University of Helsinki Finland

Academic dissertation

To be presented, with the permission of the Faculty of Biosciences of the University of Helsinki, for public criticism in Auditorium 2, Viikin Infokeskus Korona, Viikinkaari 11 on 26th September 2009, at 10 a.m.

Helsinki 2009

© Lena Alena Brüstle (Summary, Chapter IV) © Pearson Education Inc, Benjamin Cummings (Figure 1) © University of Helsinki (Appendix I) © Entomological Society of Finland (Chapter I) © Wiley-Blackwell, John Wiley & Sons (Chapter II) © Willi Hennig Society (Chapter III)

Author’s address: Finnish Museum of Natural History P.O. Box 17 (P. Rautatiekatu 13) FIN-00014 University of Helsinki Finland

Author’s email: [email protected] [email protected]

ISBN 978-952-92-6040-9 (paperback) ISBN 978-952-10-5700-7 (PDF) http://ethesis.helsinki.fi

Yliopistopaino Helsinki 2009

Die Kultur hat in Verbindung mit einer ewigen Evolution, in Jahrmillionen, aus dem Tier den Menschen geformt.

Die Zivilisation macht in wenigen Jahren mit Hilfe der Technik und Kriegsmaschinen aus dem Menschen wieder ein Tier.

Wilhelm Brüstle (1965)

Mutations as molecular tools: The metabolic-rate dependent molecular clock and DNA barcoding of allied species

Lena Alena Brüstle

List of original articles

This thesis is based on the following articles, which are referred to in the text by their Roman numerals:

I. Muona, J., and Brüstle, L. (2008) Observations on the biology of Hylochares cruentatus (Gyllenhal) (Coleoptera: Eucnemidae). Entomol. Fennica 19: 151-158.

II. Brüstle, L., and Muona, J. Life-history studies versus genetic markers - the case of Hylochares cruentatus (Coleoptera, Eucnemidae). J. Zool. Syst. Evol. Res. (in press).

III. Brüstle, L., Alaruikka, D., Muona, J., and Teräväinen, M. The phylogeny of the Pantropical genus Arrhipis Bonvouloir (Coleoptera, Eucnemidae). Cladistics (in press).

IV. Brüstle, L., Muona, J., and Salamin, N. Influence of temperature on invertebrate mutation rates: do different approaches tell the same story? (manuscript).

Table of contributions

The following table highlights the major contributions of authors to the presented articles and manuscripts listed below

I II III IV

Original Idea JM LB, JM LB, JM LB

Data Collection LB, JM LB, JM JM LB, JM

Molecular Data - LB LB, MT LB

Morphology JM JM JM, DA -

Analysis LB, JM LB, JM LB, JM LB, NS

Manuscript preparation LB, JM LB, JM LB, JM LB

LB= Lena Brüstle, JM= Jyrki Muona, NS= Nicolas Salamin, MT= Marianna Teräväinen, DA= Diane Alaruikka

Supervised by: Dr. Jyrki Muona Finnish Museum of Natural History Helsinki, Finland

Reviewed by: Prof. Jaakko Hyvönen University of Helsinki Helsinki, Finland

Dr. Ilari Sääksjärvi University of Turku Turku, Finland

Examined by: Prefekt Mari Källersjö Göteborgs botaniska trädgård Göteborg, Sweden

Contents Abstract ...... 8 I. Introduction ...... 9 DNA the Building Block of Life ...... 9 The Molecular Clock ...... 10 Concept and Application of the Molecular Clock ...... 10 Problems with the Concept of the Molecular Clock ...... 10 “Relaxing” the Molecular Clock ...... 11 The Metabolic-Rate Dependent Molecular Clock ...... 12 DNA Barcoding ...... 13 The Concept of DNA Barcoding ...... 13 Criticism of DNA Barcoding ...... 13 DNA Barcoding and Conservation ...... 14 The Molecular Clock and DNA Barcoding ...... 15 Different Purpose, similar Problem ...... 15 II. Aim of the Thesis ...... 15 III. Study Organisms ...... 16 Molecular Clock Study ...... 16 False-click beetles (Coleoptera: Eucnemidae) ...... 16 Tribe Syrphini (Diptera: Syrphidae) ...... 17 Barcoding Study ...... 17 Hylochares (Coleoptera: Eucnemidae) ...... 17 IV. Materials and Methods ...... 18 Molecular Clock Study ...... 18 Molecular and Morphological Data ...... 18 Phylogenetic Inference...... 18 Dating, Molecular Clock Models and Temperature Estimates ...... 19 Methods applied to test the Metabolic-Rate Dependent Molecular Clock .. 22 Barcoding Study ...... 23 Molecular, Morphological and Life-History Data ...... 23 V. Results ...... 23 Molecular Clock Study ...... 23 Phylogeny of the Pantropical Genus Arrhipis ...... 23 Temperature Effect on the Metabolic-Rate Dependent Molecular Clock ... 24

Barcoding Study ...... 26 Biology of the Finnish Hylochares cruentatus (Coleoptera: Eucnemidae) . 26 Hylochares cruentatus: Life-History versus Genetic Markers ...... 27 VI. Discussion and Conclusion...... 28 Discussion ...... 28 Molecular Clock Study ...... 28 Barcoding Study ...... 30 Conclusion ...... 31 Appendix I ...... 33 Appendix II ...... 34 Acknowledgements ...... 35 References ...... 37

Abstract

Mutation and recombination are the fundamental processes leading to genetic variation in natural populations. This variation forms the raw material for evolution through natural selection and drift. Therefore, studying mutation rates may reveal information about evolutionary histories as well as phylogenetic interrelationships of organisms. In this thesis two molecular tools, DNA barcoding and the molecular clock were examined. In the first part, the efficiency of mutations to delineate closely related species was tested and the implications for conservation practices were assessed. The second part investigated the proposition that a constant mutation rate exists within invertebrates, in form of a metabolic-rate dependent molecular clock, which can be applied to accurately date speciation events. DNA barcoding aspires to be an efficient technique to not only distinguish between species but also reveal population-level variation solely relying on mutations found on a short stretch of a single gene. In this thesis barcoding was applied to discriminate between Hylochares populations from Russian Karelia and new Hylochares findings from the greater Helsinki region in Finland. Although barcoding failed to delineate the two reproductively isolated groups, their distinct morphological features and differing life-history traits led to their classification as two closely related, although separate species. The lack of genetic differentiation appears to be due to a recent divergence event not yet reflected in the beetles’ molecular make-up. Thus, the Russian Hylochares was described as a new species. The Finnish species, previously considered as locally extinct, was recognized as endangered. Even if, due to their identical genetic make-up, the populations had been regarded as conspecific, conservation strategies based on prior knowledge from Russia would not have guaranteed the survival of the Finnish beetle. Therefore, new conservation actions based on detailed studies of the biology and life-history of the Finnish Hylochares were conducted to protect this endemic rarity in Finland. The idea behind the strict molecular clock is that mutation rates are constant over evolutionary time and may thus be used to infer species divergence dates. However, one of the most recent theories argues that a strict clock does not “tick” per unit of time but that it has a constant substitution rate per unit of mass-specific metabolic energy. Therefore, according to this hypothesis, molecular clocks have to be recalibrated taking body size and temperature into account. This thesis tested the temperature effect on mutation rates in equally sized invertebrates. For the first dataset (family Eucnemidae, Coleoptera) the phylogenetic interrelationships and evolutionary history of the genus Arrhipis had to be inferred before the influence of temperature on substitution rates could be studied. Further, a second, larger invertebrate dataset (family Syrphidae, Diptera) was employed. Several methodological approaches, a number of genes and multiple molecular clock models revealed that there was no consistent relationship between temperature and mutation rate for the taxa under study. Thus, the body size effect, observed in vertebrates but controversial for invertebrates, rather than temperature may be the underlying driving force behind the metabolic-rate dependent molecular clock. Therefore, the metabolic-rate dependent molecular clock does not hold for the here studied invertebrate groups. This thesis emphasizes that molecular techniques relying on mutation rates have to be applied with caution. Whereas they may work satisfactorily under certain conditions for specific taxa, they may fail for others. The molecular clock as well as DNA barcoding should incorporate all the information and data available to obtain comprehensive estimations of the existing biodiversity and its evolutionary history.

I. Introduction

And there on the side I can remember very clearly was this small model with plates for the bases - the original model with everything screwed together. And I could see the double helix! So that's when I saw the DNA model for the first time [...] and that's when I saw that this was it. And in a flash you just knew that this was very fundamental.

Nobel laureate Sydney Brenner

DNA the Building Block of Life the DNA reading frame (Lewin, 2002; Deoxyribonucleic acid (DNA) is the Freeland, 2005). genetic blueprint that determines almost all Changes on the DNA may either be characters of every known living cellular synonymous or non-synonymous. Whereas organism and most viruses (Lewin, 2002). the former “silent” mutations do not Mutations in the nucleotide sequence and change the amino acid sequence of a recombinations are the raw material for protein, non-synonymous alterations lead genetic variation and thus the mechanism to the coding of a different amino acid for the process of evolution driven by (missense mutations) or a stop codon natural selection and genetic drift. (nonsense mutations) (Freeland, 2005). Mutations mostly arise due to copying Thus, these changes alter the function of errors during DNA replication, the process the particular stretch of DNA. If non- fundamental to biological inheritance. synonymous mutations are deleterious, Several types of mutations can occur on a they may be eliminated and lost from the single gene. The replacement of one population over time. Advantageous nucleotide with another may result in two mutations, which increasing the fitness of types of nucleotide substitutions: the individual, will accrue in the transitions (changes between either purines population and result in adaptive or pyrimidines) or transversions (where a evolutionary change which may lead to purine is replaced by a pyrimidine or vice speciation events. versa). Apart from these point mutations, Non-synonymous changes may but not insertions and deletions of one or more always have to show such a profound nucleotides in the DNA sequence can effect. The replacement of a nucleotide can occur. These so called “indels” may cause result in a “neutral” or “nearly neutral” splice site mutations or lead to changes in mutation. In this case a different amino acid is encoded, but this change has no or

9 only a negligible impact on the organism’s inconstancy of mtDNA render barcoding fitness and therefore is not subject to unreliable (Hurst and Jiggins, 2005). natural selection. Several authors claim that the vast majority of mutations are neutral or at least nearly so (Kimura, 1980; The Molecular Clock Ohta, 1987; Ohta, 2002). Concept and Application of the Molecular The neutral theory of molecular evolution, Clock yet heatedly debated (for a review of the The concept of a molecular clock has been “neutralist-selectionist controversy” see first described by Zuckerkandl and Pauling Kimura, 1993; Nei, 2005), is fundamental (1962) on the basis of the recognition of for the molecular clock concept rate replacement uniformity in the α-globin (Zuckerkandl and Pauling, 1962). The gene. Further studies have shown the molecular clock hypothesis predicts that average substitution rate per site per year mutation rates are proportional to to be 10-9 across several proteins of evolutionary time and thus molecular different species, indicating relatively differences between species can reveal constant rates of molecular evolution their time of divergence. (Kumar and Subramanian, 2002). The Mutations are further employed to identify discovery that substitution rates of organisms via “genetic barcodes” nucleotides in DNA and RNA and (Kurtzmann, 1984). DNA barcoding aims consequently of proteins are proportional to effectively delineate organisms to to evolutionary time, has led to the belief species-level relying only on genetic that the average divergence time of taxa variation found in a short region of a could be calculated. This idea of a protein-coding mitochondrial gene (Hebert molecular clock is still embraced by many et al., 2003). The significance of neutrality scientists, but fundamental problems for DNA barcoding is controversial and it regarding the assumptions of the molecular is unclear whether mitochondrial DNA clock exist. (mtDNA) is truly neutral: Some have criticized the usage of this neutral locus, not causally related to fitness, to delineate Problems with the Concept of the taxa (Matthew et al., 2008). Others have Molecular Clock argued that the non-neutrality and The main assumption underlying the molecular clock is its neutrality, which

10 implies that molecular differences do not the effectiveness of error correcting affect the fitness of organisms. Thus, the polymerases and patterns of inheritance dynamics of present changes in a may also play a role (Rodriguez-Trelles et population are believed to be determined al., 2004; Wolfe et al., 1987). Even by random genetic drift. According to the seemingly constant rates might only be an neutral theory most of the observed artefact of the molecular test applied, since polymorphisms should therefore be most tests used to identify and exclude selectively neutral (Kimura, 1968). The sequences that violate rate-constancy neutral molecular clock is predicted to be assumptions only show limited statistical stochastic, following a simple Poisson power (Dobzhansky et al., 1977; Scherer, distribution of substitution occurrences 1989). For example evolutionary rate (Hartl and Clark, 1997). Thus, the mutation differences among lineages may not be rate should be constant over time. revealed using standard molecular clock tests for common alignment length A growing number of molecular studies (Rodriguez-Trelles et al., 2004). have revealed that the assumption of neutrality does not always hold, challenging the existence of a universal, “Relaxing” the Molecular Clock uniform mutation rate. Rather, variances in molecular clock rates have been observed The molecular clock is increasingly being (Pawlowski and Berney, 2004) between used (e.g. Knapp et al., 2005; Renner, lineages (Britten, 1986), different types of 2005), but not without trying to account for DNA, types of mutations (Wolfe et al., some of its problems. Hypotheses have 1987), and even within different regions of been formulated to explain the protein-coding, previously assumed discrepancies between observed rates and neutral, mitochondrial genes (Ballard and those predicted by the strict molecular Whitlock, 2004). clock. New “relaxed molecular clock” approaches have been developed. Relaxed Several reasons can explain the erratic clocks do not assume the biologically behaviour of substitution rates: Life- unverified hypothesis of a constant history traits such as generation time, body evolutionary rate over time but take the size, body temperature, effective heterogeneity of substitution rates into population size and changes in the account (Douzery et al., 2004; Rodriguez- environment can have an effect on Trelles et al., 2004; Renner, 2005). Many mutation rates. “Biological properties” like different relaxed clock models exist such

11 as local clocks (Yoder and Yang, 2000), Expressed as a formula: episodic clocks (Gillespie, 1991), -1/4 -E/kT autocorrelated clocks (Sanderson, 1997; B = bo M e Sanderson, 2002) or uncorrelated relaxed clocks (Drummond et al., 2006). Where B stands for mass-specific

metabolic rate, bo is a coefficient A broad consensus has been reached that independent of body size and temperature, these relaxed clock models yield better M-1/4 is the body size “quarter-power- results than strict clocks and thus have average” and e-E/kT is the Boltzmann factor become popular tools to date speciation (for details see Gillooly et al., 2005). events. Since its proposition, this model has been

tested further (e.g. Thomas et al., 2006; The Metabolic-Rate Dependent Molecular Estabrook et al., 2007; Lanfear et al., Clock 2007) and the idea of a metabolic-rate dependent molecular clock (hereafter Despite the skepticism regarding a strict metabolic clock) has been accepted molecular clock and the advances to (Estabrook et al., 2007) as well as rejected improve dating through applying relaxed (Thomas et al., 2006; Lanfear et al., 2007). clock models, Gillooly et al. (2005) Some of these studies have been criticized recently proposed a model for animals (Mittelbach et al., 2007) for only which suggests that: correcting for body size but ignoring “…there is indeed a single molecular clock, as temperature (e.g. Thomas et al., 2006) and originally proposed by Zuckerkandl and Pauling primarily focusing on mammals (e.g. [Zuckerkandl, E. & Pauling, L. (1965) in Evolving Gillooly et al., 2005) thus not revealing a Genes and Proteins, eds. Bryson, V.& Vogel, H. J. (Academic, New York), pp. 97–166], but that it universal metabolic rate effect also valid „„ticks‟‟ at a constant substitution rate per unit of for invertebrates (Thomas et al., 2006; mass-specific metabolic energy rather than per unit Lanfear et al., 2007). of time. This model therefore links energy flux and genetic change. More generally, the model suggests The contradicting conclusions regarding that body size and temperature combine to control the metabolic clock and shortcomings of the overall rate of evolution through their effects on previous work have led to a debate on metabolism.” whether such a clock exists. Further studies

need to be carried out to shed more light on

the universality of a metabolic clock.

DNA Barcoding DNA barcoding aspires to be a standardized, cost- and time-effective The Concept of DNA Barcoding technique to assign organisms to lower The first 648 base pairs of the 5’ region of taxonomic categories solely relying on a the mitochondrial gene cytochrome c short stretch of a single DNA sequence. oxidase subunit I (COI) have been The underlying assumption of this method proposed as a genetic “barcode” for is that intraspecific variation found in the animals. The DNA barcode has been barcode region is considerably smaller praised by its advocates as the solution to a (<3%) than interspecific variation (>3%) collapsing taxonomic work-force (Hebert (Hebert et al., 2003) and that it is on et al., 2003). Inadequate numbers of average 10x lower within than between qualified taxonomists, limitations of the species of the group under study. This morphologically-based identification threshold is also known as the “barcoding system (Hebert et al., 2003; Waugh, 2007), gap” (Hebert et al., 2004). Barcoding poor knowledge of species diversity advocates claim that, at least for animals, (Rubinoff et al., 2006) and increased the nucleotide substitution rate of COI is threats to the earth’s ecosystems have high enough to distinguish not only prompted a call for a more efficient between closely related species but also approach to catalog the world’s between phylogeographic groups within a biodiversity, such as barcoding (Blaxter, single species (Hebert et al., 2003). Thus, 2004; Smith et al., 2008). However, the main advantages of barcoding are its barcodes are neither regarded as the only speed and accuracy compared to labour solution nor the sole attempt to overcome intensive, traditional taxonomy. the present taxonomic shortcomings. Thus, the Convention of Biological Diversity (CBD) recognizes taxonomic knowledge Criticism of DNA Barcoding as a key input in the management of all Critics of barcoding have argued that the kind of ecosystems. Therefore, the CBD “application of a quick-fix, automated- seeks to implement action plans for pragmatist model is antithetical to a taxonomic capacity-building and aims to science endowed with a strong double the taxonomic workforce by 2020 epistemological and theoretical as part of the Global Taxonomy Initiative foundation” (de Carvalho et al., 2008) and (GTI) (Convention of Biological Diversity, doubts regarding this method exist: It has 2006). been questioned whether a short stretch of

13 mtDNA such as the COI barcoding region barcoding, next to nothing about its value, can show enough resolution to detect the biological importance (Rubinoff et al., enormous number of species that are 2006) or best conservation practices will supposed to be identified applying this be known. approach (DeSalle et al., 2005). Especially since inheritance of the mitochondrial genome is not always predictable due to DNA Barcoding and Conservation e.g. heteroplasmy (Gryzbowski et al., Especially in conservation biology DNA 2003), recombination (Tsaousis et al., barcoding can be seen as a hindrance rather 2005) or exceedingly common Wolbachia than a blessing. If species identification is infections in insects (Whitworth et al., based on the barcode only, biologists 2007). Indeed, barcoding has been shown ignorant of most other characters will have to fail to distinguish between closely to justify conservation actions solely based related or morphologically very similar on a small portion of one genome species (Armstrong and Ball, 2005; (Rubinoff, 2006). Hajibabaei et al., 2006; Meier et al., 2006). The fact that the evolution of species is a Even if COI contained enough information continuous and dynamic process whereas to reliably determine species, a universal barcoding is a “yes” or “no” identification cut-off level for inferring species status method, leads to following questions if does not exist, since intra- and interspecific conservation is wholly based on DNA: genetic distances have shown to overlap considerably (Goldstein et al., 2000; 1) How can scientists convince the public Wiemers and Fiedler, 2007; Jansen et al., to conserve otherwise similar species just 2009) and thus cut-off points will have to because they differ in barcodes, a concept be continuously revised from group to they do not comprehend? group (DeSalle et al., 2005). Moreover, the 2) And should then, as a logical DNA barcode species concept is just consequence, populations varying in another addition to many occasionally morphology and life-history but sharing contradicting species concepts, with none identical mtDNA be allowed to go extinct being able to declare precedence over the (Rubinoff, 2006)? others (Coyne and Orr, 2004). To circumvent these limitations of a If despite these shortcomings a species has simplified (and thus appealing) approach been identified solely on the basis of DNA such as DNA barcoding it has been

14 suggested that large-scale sequence certain taxa, since too fast as well as too datasets should be combined with all of the slow mutations can lead to lack of other available data in order to create as resolving power of COI-sequences (Frézal comprehensive estimations of the existing and Leblois, 2008). biodiversity as possible (Smith et al., If species will be identified solely on the 2006). basis of the DNA species concept, DNA barcoding will become a prerequisite for estimating speciation dates. If then The Molecular Clock and DNA inaccurate mutation rates are calculated Barcoding because of the shortcomings of DNA Different Purpose, similar Problem barcoding and the, to a certain extent, subjectivity of sequence alignments DNA barcoding and the molecular clock (Wheeler, 1996) these incorrect rates will are both tools in molecular biology applied be compounded in the molecular clock. to serve different purposes. Whereas DNA Thus, despite the two methods serving barcoding primarily seeks to identify and different purposes, their common reliance delimitate species, the molecular clock on mutation rates renders them vulnerable calibrates divergence times between to the same errors. already established phylogenetic groupings. DNA barcoding and the molecular clock are both based on the II. Aim of the Thesis same principle of countable mutations accumulating over evolutionary time. This thesis aims to examine the Molecular clock studies have shown that effectiveness of mutations as molecular mutation rates often are neither neutral, tools. Two aspects of widely applied, yet constant nor uniform even if the same gene controversial molecular methods, DNA is compared across organisms. This barcoding and the molecular clock were observation has not only weakened the tested: molecular clock theory but has also posed problems to DNA barcoding: The success The first part of the thesis investigated the of barcoding rests upon the assumption efficiency of DNA barcoding to distinguish that COI mutates at such a rate that species reproductively isolated, endangered delimitations are feasible. “Inappropriate” invertebrate populations. This was done in mutation rates have rendered this futile for two steps. First, the life-history of the

15 organisms was thoroughly studied (Paper documented groups whose distribution and I). Insights from this study were combined phylogenies inferred from morphological with morphological observations and characters reflect a Gondwanan break-up compared to molecular data, in particular scenario (Muona, 1991; Muona, 1993). to the DNA barcoding region. Implications However, the pattern shown by most of the of the results for conservation actions were false-click beetle groups is the rather assessed (Paper II). unusual North Gondwanan Pattern (NGP) (Sanmartín and Ronquist, 2004). In the second part of the thesis the underlying temperature effect on the The genus Arrhipis Bonvouloir, a metabolic clock was tested. Again, this monophyletic Pantropical group with was done in two steps: First, the vicariant relationships (Muona, 1991) evolutionary relationships of the group seemed to be one of the taxa displaying the under study were established using NGP (Sanmartín and Ronquist, 2004). A morphology and molecular markers (Paper considerable amount of newly available III). This phylogeny was then applied as morphological material as well as one of two datasets to test whether molecular data prompted a revision of the temperature has an effect on the constancy taxon to gain a deeper understanding of its of mutation rates in invertebrates of equal evolutionary history, particularly regarding body size (Paper IV). its Gondwanan origin (Paper III).

The genus Melasis Olivier exhibits a Holartic distribution and is thus only found III. Study Organisms in temperate regions. The Scandinavian

and Eastern North American Melasis are Molecular Clock Study sister species (Muona, pers. comm.) and

False-click beetles (Coleoptera: their phylogeny corresponds to the Eucnemidae) vicariant break-up pattern of the Laurasian northern trans-Atlantic connection False-click beetles are usually small- to (Sanmartín et al., 2001). The split between medium-sized polyphagan beetles, and are North America and Europe was used to distributed worldwide in six date the Arrhipis/Melasis tree to compare biogeographical regions (Nearctic, temperate and tropical mutation rates Palaearctic, Neotropical, African, Oriental (Paper IV). and Australian) (Alaruikka, 2004). Eucnemidae are one of the best

Arrhipis and Melasis live in rotten tree Barcoding Study trunks and are poor dispersers. They are Hylochares (Coleoptera: Eucnemidae) similar in body size and have a generation time of one generation every two years Hylochares cruentatus Gyllenhal (Col., (Muona, pers. obs.; Muona, 1993). Eucnemidae) is a scarce species with a limited range in the Western Palaearctic. It

has always been reported as being very Tribe Syrphini (Diptera: Syrphidae) rare in Finland with the last sighting dating back to probably the 1920s (Muona, 1984). Hoverflies (Syrphidae) comprise 6000 Thus, it has been thought to have gone described species in 14 tribes and 3 locally extinct in the last century (Siitonen presently recognized subfamilies and Martikainen, 1994) possibly due to (Thompson and Rotheray, 1998; forestry practices (Rassi et al., 2001) in Thompson, 2006). particular the disappearance of aspen The tribe Syrphini consists of 42 genera (Populus tremula L.). which are classified into 59 subgenera and Unexpectedly, a single specimen was are present world-wide. The phylogeny of captured with a pit-fall trap in Vantaa, 27 genera representing 33 subgenera has Southern Finland in 2004 (Nieminen et al., recently been established based on 2008 ). Although studies in Russian Karelia molecular data (Mengual et al., 2008). Due found the beetle to be a specialist tightly to their wide distribution, Syrphini are bound to large, dead aspen trees (Kangas found in different temperature regimes, and Kangas, 1944; Siitonen and with even sister species living in Martikainen, 1994; Siitonen et al., 1996) remarkably different temperature the Finnish specimen inhabited a willow conditions (Paper IV). Syrphini have (Muona et al., 2008). From this finding it between one and two generations per year appeared that the presence of over-aged (Keil et al., 2008) and have similar body surface-rotten aspens is not the crucial sizes (Ståhls, pers. comm.). Due to their aspect for the survival of H. cruentatus. different temperature regimes, similar life- The question arose whether this biological history traits and relatively large species difference between the Russian and numbers, the tribe Syrphini was an ideal Finnish beetles is due to: candidate to complement the Eucnemidae dataset when testing the temperature a) an underestimated ecological plasticity effects on a metabolic clock (Paper IV). b) the taxa actually not being conspecific

To answer this, further investigations into This is due to mitochondria being the the life-history traits of the Finnish (Paper production sites of reactive oxygen species I) as well as the genetic make-up of the (ROS) which are toxic by-products of Finnish and Russian populations (Paper II) metabolism. Therefore, DNA damage from were carried out. metabolites might be higher and thus mutation rates faster in mitochondrial

rather than nuclear genes, especially in IV. Materials and Methods warmer climates (Martin and Palumbi, 1993; Fontanillas et al., 2007). Molecular Clock Study For the Syrphidae dataset DNA sequences

Molecular and Morphological Data for 1) the mitochondrial Cytochrome c Oxidase subunit I (COI) and 2) the nuclear DNA was extracted from the Arrhipis and large-subunit ribosomal 28S gene were Melasis species plus the outgroups. Due to obtained from Genbank the poor quality of the DNA retrieved from (http://www.ncbi.nlm.nih.gov/) (Paper IV). the dried and pinned museum specimens Eucnemid morphological data (Alaruikka five genetic markers were chosen on the and Muona, unpublished) was revised and basis of most successful DNA additional genera were added to the matrix amplification. The following four loci from in order to analyse the position of Arrhipis. the mitochondrial genome and one nuclear Twenty-six morphological characters, gene were used (Paper III & IV). some of them displaying multiple states (a Mitochondrial: 1) partial sequence of the total of 49 binary characters), were small subunit ribosomal 12S gene, 2) analysed (Paper III). partial sequence of the large-subunit ribosomal 16S gene, 3) partial sequence of the Cytochrome c Oxidase subunit I (COI) Phylogenetic Inference and 4) partial sequence of the Cytochrome b (Cytb) gene. Nuclear: 1) small-subunit A phylogenetic analysis combining ribosomal 18S gene. For testing the morphology as well as molecular data was temperature effect of the metabolic clock performed for the Arrhipis dataset using (Paper IV) it was useful to sequence parsimony as the optimality criterion (POY mitochondrial as well as nuclear genes. It version 3.011.a (Wheeler, 1996; Giribet et has been argued that the metabolic rate al., 2002)). Additionally, a Bayesian effect acts much stronger on the former. inference analysis on the Arrhipis

18 molecular data was made (MrBayes 3.1.2 analyse the data increased the confidence (Ronquist and Huelsenbeck, 2003)) (Paper in the results. III). The underlying principle of the use of To the obtained topology (Paper III) the parsimony as an optimality criterion for species of the genus Melasis were added as reconstructing phylogeny is to assume as the sister group of the genus Arrhipis little as possible about any mechanism of according to their relationship described by evolution. Each evolutionary event is Muona (1993) (Paper IV). To make sure unique and consequently no a priori the placement was correct a BEAST chosen model for species formation can be (Bayesian Evolutionary Analysis Sampling applied. Therefore, instead of relying on a Trees) (Drummond and Rambaut, 2007) statistical framework to find the “best” analysis was carried out on the Arrhipis topology, parsimony favours the tree that and Melasis molecular data. BEAST requires the fewest evolutionary changes allowed the constraining of the Arrhipis (Steel and Penny, 2000). Thus, the clade to the relationships obtained in Paper explanatory power of a phylogeny depends III. The topology for the tribe Syrphini was on the degree to which it can minimize taken from the recently published homoplasies (characters whose origin can parsimony tree of predatory flower flies not be explained by common ancestry) (Diptera, Syrphidae, Syrphinae) (Mengual (Farris, 1983). In contrast, the Bayesian et al., 2008). The Eucnemidae and the approach is a statistical inference using Syrphidae tree were then used to test the Markov chain Monte Carlo (MCMC) prediction that taxa in colder climates have methods to obtain the “best” tree. It allows lower mutation rates, due to slower the choice of a specific model of evolution biochemical processes, than those taxa as well as the incorporation of any found in warmer climates (Paper IV). available prior information. The posterior probability, which is proportional to the product of the likelihood of the data given Dating, Molecular Clock Models and the model and the prior probability, is then Temperature Estimates calculated. The phylogenetic tree with the highest posterior probability is favoured as The split of Pangea into the two super- the most likely one (Felsenstein, 2004). continents Gondwana and Laurasia and the Using such philosophically and subsequent break-up of first Gondwana methodologically differing approaches as and then Laurasia is a firmly accepted parsimony and the Bayesian framework to concept today (Li and Powell, 1993;

Sanmartín et al., 2001) (Fig. 1). In Melasis species via this landmass were too biogeography the age of taxa has been harsh (Muona, pers. comm.). correlated with the age of geographic events, since it has been concluded that earth and life evolved together, during phases of uplift, continental drift and many other processes. Thus, biogeography so far seems to be the most promising method in dating evolution, since it clearly distinguishes age of being and age of fossilization (Heads, 2005).

The phylogenies of the Arrhipis and

Melasis genera show a vicariant Gondwanan and Laurasian break-up pattern, respectively. Speciation events in the genus Arrhipis reflect the opening of the South Atlantic Ocean causing Africa to split from South America around 120-100

million years ago (mya) (Paper III, Murphy Fig. 1. Break-up of Pangea et al., 2001; Sanmartín and Ronquist,

2004). The Eucnemidae topology was dated using the Melasis North America and Assuming that mutation rates from Europe split (Paper IV, Study I). These two organisms found in temperate regions are landmasses are believed to have separated slower than those from tropical organisms in the Early Tertiary, 65-55mya (Janis, (Gillooly et al., 2005) we predicted that 1993; Sanmartin et al., 2001). Even though dating our topology with the ~60mya a land bridge connecting North America Melasis split will show older speciation and Scandinavia existed until around events within the genus Arrhipis than 40mya (Sanmartín et al., 2001), the would be expected from the tectonic ~120- dispersal of the Melasis via this route is 100mya Gondwanan break-up date (Paper highly unlikely, since the movement was IV, Study I). restricted to very cold adapted organisms. To see which gene fits which molecular Thus, conditions for the dispersion of the clock model best, likelihood ratio tests

20 were carried out using BASEML in the using the WorldClim database in ArcMap PAML v3.15 package (Yang, 1997). Strict (http://www.esri.com/software/arcgis/). clock (one constant rate assumption), local These temperature estimates were then clock (the global clock rate is divided into transformed into the Boltzmann factor, several, local rates), autocorrelated clock which underlies the temperature (autocorrelation puts a limit on the speed a dependence of metabolic rate: rate is allowed to change from the ancestor to the descendant) and no-clock (no rate Boltzmann factor = e-E/kT assumption) models were tested. The clock model with the highest likelihood score Where E is an average activation energy plus the no-clock model were used to for the biochemical reaction of metabolism obtain substitution rates and branch length (~0.65 eV), k is the Boltzmann’s constant for each gene separately. Each tree for (8.62 x 10-5 eV.K-1) and T equals absolute every gene was fixed according to the temperature in degrees Kelvin (Gillooly et Arrhipis/Melasis topology (Fig. 3) and the al., 2001; Gillooly et al., 2005). trees were dated using the appropriate The calculated Boltzmann factors as well clock models in BASEML and as temperature estimates in degrees were MULTIDIVTIME (Yang, 1997; Thorne used as surrogates for body temperature and Kishino, 2002) (Paper IV, Study I). (Paper IV: Boltzmann factor used in Study For the Syrphidae dataset no divergence I + III, temperature estimates together with time calibration points were available, thus average number of frost free days per the phylogenetic tree (obtained from annum applied in Study II, according to Mengual et al., 2008) could not be dated Estabrook et al., 2007) since it was and therefore only branch lengths under a assumed that “extant ectotherms are no-clock model for each gene individually approximately in thermal equilibrium with were acquired in BASEML (Yang, 1997) their environment, and that they occur in a (Paper IV, Study II+III). similar thermal environment as their Longitude and latitude coordinates for the ancestors” (Muona, 1993; Gillooly et al., collection sites of each species were 2005). obtained from mapping the collection location in Google Earth (earth.google.com). Average yearly temperatures and number of frost free days for each data-point were then acquired

Methods applied to test the Metabolic-Rate was done to see whether correlations Dependent Molecular Clock between the Boltzmann factor and substitution rate and the Boltzmann factor Three methods were chosen to test the and branch length are present across the temperature effect on a body size corrected tree (Paper IV, Study I). metabolic clock (Paper IV). In previous studies these methods had revealed a The second analysis was carried out temperature and body size effect (Gillooly according to Estabrook et al. (2007). The et al., 2005; Estabrook et al., 2007) or at Eucnemidae and Syrphidae datasets were least a body size effect (Fontanillas et al., used. Branch length (accumulated genetic 2007) on the metabolic clock: change since the most recent common ancestor) and temperature conditions were The first study (Paper IV) was carried out compared within species pairs across the according to Gillooly et al. (2005) and the tree as well as within sister pairs. The aim Eucnemidae dataset was used. Mutation was to calculate the number of “monotone rates of temperate and tropical species pairs”, where the most genetically were studied across the tree. The topology differentiated species of a pair also exhibits was dated for each gene using the a faster metabolic rate. This was done molecular clock model of best fit. The no- using a programme called ECERFODM. clock model was also applied. The aim was ECERFODM calculates the thermal to infer whether: regimes for each ancestral node by taking a) the temperate clade mutates slower than the temperature average obtained from the the tropical one and whether results are immediate descendants (Paper IV, Study consistent, regardless of the choice of II). clock models or mode of expressing The third approach was based on genetic change used Fontanillas et al. (2007). The Syrphidae b) correcting for temperature will reconcile dataset was analysed. Sister pairs were molecular and biogeographical divergence studied as well as independent pairwise dates and lead to a strict molecular clock comparisons were carried out. This was done to see whether those species with Two-tailed non-parametric Spearman’s longer branch length and thus more rank correlation tests were performed. The mutations, also live in warmer climates Boltzmann factor for ancestral nodes was (expressed as the Boltzmann factor). For reconstructed using “ace” in the R package the comparisons between sister species the “ape” (http://www.R-project.org). This

22 relative biological trait variable (the ratio amplified since the primers (Navajas et al., of the Boltzmann factor of the species with 1998) seem not to be specific enough for the higher temperature over the species Eucnemidae (Teräväinen, pers. comm.). with the lower temperature) and relative Obtained DNA sequences for each gene substitution rate (the ratio of the branch were aligned in ClustalW2 (Larkin et al., length of the species with the higher 2007) and checked for genetic differences temperature over the branch length of the between the Finnish and Russian species with the lower temperature) was populations (Paper II). calculated. Then two-tailed non-parametric Actual observations of the Finnish beetles Spearmann’s rank correlation tests were at their sites of occurrence in the field were performed. Independent pairwise made over a time-span of two years (2006- comparisons were carried out using 2008). Behaviour, life-history traits as well Mesquite version 2.5 (Maddison and as habitat characteristics were recorded Maddison, 2008) (Paper IV, Study III). (Paper I) and compared with the information available from the Russian populations (Kangas and Kangas, 1944; Barcoding Study Siitonen and Martikainen, 1994; Siitonen Molecular, Morphological and Life- et al., 1996). Larval features and larval History Data galleries as well as morphological characters of adult male and female DNA was extracted from Finnish and Finnish and Russian specimens were Russian Hylochares specimen. Two studied and compared (Paper II). mitochondrial and two nuclear genes were selected. Mitochondrial: 1) partial sequence of the small-subunit ribosomal V. Results 12S gene and 2) partial sequence of the

Cytochrome c Oxidase subunit I gene Molecular Clock Study (COI) (barcoding region). Nuclear: 1) small-subunit ribosomal 18S gene and 2) Phylogeny of the Pantropical Genus partial sequence of the large-subunit Arrhipis ribosomal 28S gene. Unfortunately the No conflicting results were observed second internal transcribed spacer (ITS2), between the combined analyses of due to its high mutation rate useful in morphological and molecular data using species delimitation, could not be equal weights, gap cost 2 or gap cost 4 in

POY. The single parsimonious tree (length The retrieved Bayesian tree only differed 1978 steps) found with the equally from the parsimony tree for two taxa: weighted analysis (Fig. 2a) shows one of Protofarsus and A. gaillardi (Fig. 2b). the possible fully resolved trees of the POY places A. gaillardi within the African consensus solution obtained with gap cost clade and Protofarsus between the 2 and gap cost 4. outgroup Microrhagus and Arrhipis. MrBayes though groups A. gaillardi and

2a. Protofarsus as sister to the American clade although the support for this placement is very low (posterior probability = 0.55). Thus, the maximum parsimony phylogeny inferred with the equal weight solution gives a well supported hypothesis for the data (Fig. 2a) (Paper III).

Temperature Effect on the Metabolic-Rate Dependent Molecular Clock

2b. In Paper IV the Eucnemidae dataset (Fig. 3) was applied in Study I & II and the Syrphidae dataset (Fig. 4) in Study II & III. No significant underlying temperature effect on mutation rates could be found in any of the three methodological approaches for either dataset.

In Study I (according to Gillooly et al., 2005) only 2 out of 12 correlation tests showed a significant relationship between

temperature and mutation rate. These two Fig. 2a. POY Arrhipis combined analysis Fig. 2b. MrBayes Arrhipis combined analysis positive correlations were furthermore

Colours in Fig. 2a. & b. show geographic regions found only for one out of the five genes Green= Asia, Red= Africa, Blue= Asia, Orange= Australia, Grey= Outgroups studied: Cytb for branch length when Values in Fig. 2b are posterior probabilities

applying the no-clock model (p= 0.02) and

Cytb for branch length under the local mutation rate was found for either 28S or clock model (p= 0.01) (but not for COI when comparing sister species or temperature and substitution rate using a when carrying out independent pairwise local clock (p= 0.53)). Cytb was also one comparisons. of the only two genes (the other one being To summarize, none of the studied genes 16S) which, when dated with the ~60mya (mitochondrial, nuclear) in any of the two split, showed an older (~400mya instead of invertebrate datasets (Eucnemidae, ~120-100mya) South American/African Syrphidae) showed a consistent significant split than assumed from tectonic break-up correlation between temperature and events. Thus, only Cytb exhibits too old mutation rate. This was the case, regardless tropical speciation dates combined with a of the used clock models (strict, local, significantly positive correlation between autocorrelated or no-clock), mode of temperature and mutation rate, as predicted expressing genetic change (substitution by Gillooly et al. (2005). Therefore, rate, branch length) temperature estimates correcting for temperature was abandoned, (Boltzmann factor, degrees) or since it would have neither reconciled methodological approach (comparison molecular and biogeographical divergence between species pairs across the tree, sister dates nor led to a strict molecular clock for pairs, independent pairwise comparisons) most of the genes studied. (Paper IV). In Study II (according to Estabrook et al.,

2007) only 16S showed a significant (p= 0.01) number of monotone pairs over the whole phylogenetic tree. Since in this study body size was accounted for, faster metabolism can be explained with the positive effect of temperature on mutation rates. None of the other genes showed such an effect neither when analyses were carried out across the whole tree nor between sister species only.

In Study III (based on Fontanillas et al., Fig. 3. Eucnemidae phylogeny obtained from BEAST using molecular data

2007) no significant positive or negative Red= Holarctic Melasis, Black= Pantropical Arrhipis 60mya= calibration point: Europe/North America split 120-100 mya= tectonic Africa/South America split correlation between temperature and

Fig. 4. Tribe Syrphini phylogeny obtained from the direct optimization analysis using POY for molecular data (Mengual et al., 2008)

Barcoding study (Salix pentandra L. and S. myrsinifolia Salisb.) in urban forested wasteland sites in Biology of the Finnish Hylochares the Helsinki metropolitan region. No H. cruentatus (Coleoptera: Eucnemidae) cruentatus were found in Alnus spp., P.

Hylochares cruentatus (Fig. 5) has only tremula, Salix fragilis L. or Salix caprea L. two close relatives H. harmandi Fleutiaux growing at the particular site. Its favoured found in the Far-East and Japan and H. habitat is a continuum of S. pentandra and nigricorn is (Say) found in the Nearctic. S. myrfinifolia infested with the fungus Within the European Union the only Phellinus igniarius (L.) Quél. next to known extant populations of H. cruentatus regularly flooding small waters. Parts of are found in Finland. the trunk of the willow trees appear to be dead and the fungal infestation is strong. The Finnish H. cruentatus breeds in large Emergence holes in the hard and sound and partly hollow and broken willow trees

26 appearing wood were found. Female and Even though no larval features were found male beetles could be observed. Matings that allowed the separation of the Finnish and egg-laying were sighted and larvae and Russian Hylochares the two were found on several occasions (Paper I). populations differed in their adult morphology. Morphological characteristics The Finnish collections made contrasted known to be useful for separating species greatly with those by Kangas and Kangas within the Eucnemidae family include (1944) and Siitonen and Martikainen body proportions, antennal structure and (1994) which showed that the H. male genitalia. The Finnish and Russian cruentatus found in Russia live in large, Hylochares differed in the proportions of dead P. tremula with larvae not penetrating the fused lateral lobes of the aedeagus, the the hard wood at all. These observations structure of the median lobe of the from Russian Karelia have been considered aedeagus and proportions of the male and as typical for H. cruentatus. female antennomeres as well as the

structure of the hypomera. In addition to these features the shape of the pronotum

varied, although differences within populations were observed. Despite these

morphological distinctions no sequence divergence was found in any of the four analysed genes including the COI

barcoding region. Thus, genetic-makers failed to separate the two populations

clearly distinguishable by their morphological characters. Fig. 5. Previously considered locally extinct Hylochares cruentatus female on Salix In spite of their indistinguishable genetic pentandra, Finland, Vantaa make-up the two taxa are considered to

belong to two separate species. Their Hylochares cruentatus: Life-History versus identical genetic constitution can be taken Genetic Markers as:

The biology of H. cruentatus in Finland a) an indicator of a recent divergence event differed markedly from that of the Russian not yet reflected in the markers analysed Hylochares populations (Paper I).

27 b) the chosen genes falling short to VI. Discussion and Conclusion distinguish between the two closely allied Hylochares species Discussion

COI has been previously reported to fail Molecular Clock Study delimitating related species in other insect studies such as Lepidoptera (Kaila and The combined parsimony analysis using Ståhls, 2006; Wiemers and Fiedler, 2007), POY revealed that the phylogenetic Diptera (Stevens et al., 2002; Whitworth et hypothesis by Muona (1991) and the al., 2005; Meier et al., 2006) and previously suggested Northern Gondwana Hymenoptera (Quicke, 2004). Further, it Pattern (NGP) is incorrect. The new results has to be kept in mind that species are, at (with high support values) show mostly least to some extent, artificial groupings tropical monophyly of the genus Arrhipis. created by scientists and numerous species The topology is in concordance with the concepts make classifications difficult and Southern Gondwana Pattern (SGP) not always clear-cut. In this case, due to excluding New Zealand (Fig. 6). the greatly differing ecology and Since the SGP is explained by vicariance, morphology between the Finnish and the Gondwanan break-up dates are robust Russian Hylochares populations it seems calibration points for the Arrhipis appropriate to classify the non-Finnish phylogeny and thus make this genus a Hylochares as a separate species, useful candidate for testing molecular Hylochares populi. clock models as was done here (Paper IV). Two Hylochares species can be identified,

the newly described Russian H. populi and the Finnish H. cruentatus. The latter species is endemic to Finland and has only recently been re-discovered. Therefore, H. cruentatus must have high conservation

priority (Paper II). Fig. 6. The Southern Gondwana Pattern (SGP) The SGP is the most well known Gondwanan pattern. It is explained by a vicariant sequential break-up of Southern temperate Gondwana with extinctions and primitive absences (Sanmartín and Ronquist, 2004). S= Southern

No consistent metabolic rate effect on the Several methodological approaches were molecular clock was found regardless of used, resulting in comparisons across the the methodology applied or genes used. whole phylogenetic tree, independent Thus, these results confirm observations of pairs, as well as between sister species a previous invertebrate study which also only, to investigate the temperature effect found no such effect when correcting on mutation rates. Mitochondrial as well as mutation rates for size and temperature nuclear genes were studied and two (Lanfear et al., 2007). It therefore has been invertebrate datasets were analysed. argued that body size rather than Shortcomings of previous analyses, such as temperature may be the underlying force the failure to distinguish between the behind a metabolic clock (Lanfear et al., generation time and body size hypothesis 2007; Fontanillas et al., 2007 - although (Gillooly et al., 2005), were accounted for. only for mitochondrial genes). Several Despite this, some methodological studies have found a link between body drawbacks have to be mentioned: size and mutation rates in vertebrates Environmental temperatures are only (Estabrook et al., 2007) and especially in surrogates and may not always reflect mammals (Gillooly et al., 2005; Welch et actual body temperatures, a problem al., 2008). The body size effect, thus applying to other studies as well (e.g. proposed to hold for vertebrates, remains Gillooly et al., 2005; Estabrook et al., controversial for invertebrate taxa (Thomas 2007; Lanfear et al., 2007). Even though et al., 2006; Fontanillas et al., 2007; species show very similar body sizes and Lanfear et al., 2007) and does not seem to sister species are extremely close in size, be revealed in some groups unless deep overall size variations between the species comparisons across the phylogenetic tree exist. However, these differences are not are studied (Fontanillas et al., 2007). significant and thus we are confident that they did not confound the results. Here, shallow comparisons on the genus Vicariance divergence dates only and species level were carried out. Hence, approximate estimations of actual the body size effect as a driving force speciation events, and may lead to behind the metabolic clock might not be erroneous calibrations of mutation rates. present in the studied invertebrate datasets. Therefore sister pair comparisons that Therefore, the temperature effect alone, if circumvent this problem were also carried existent at all, may be too weak to have a out. Last, the Eucnemidae dataset used was statistically significant impact on mutation rates.

29 rather small. To counteract this, the larger running to the Gulf of Finland 2200 years Syrphidae dataset was employed as well. ago. The locality where this saproxylic beetle thrives in is in the middle of a city To conclude, no contradicting results and the small river is considered essential between the majority of the statistical tests, for drainage of rain water from the genes or invertebrate groups were found. surrounding regions. This, in addition to The consistency of these results gives the suitability of the area for outdoor confidence that there is no underlying activities and low housing development temperature effect on mutation rates and potential, have saved the beetle this far. that no universal strict metabolic clock is Thus, not unexpectedly, light human present in the studied taxa. influence combined with long continuous ecosystem history is a major contributing factor to the survival of this population. Barcoding Study In order to save this endemic species new The biological differences and the conservation plans have to be established classification of the Finnish and Russian as soon as possible. Some conservation Hylochares into two distinct species have actions have already been implemented: wide implications for the conservation of Finnish amateur entomologists were made H. cruentatus. aware of the novel Hylochares through a H. cruentatus was regarded as an aspen- publication in an amateur entomologist specialist and its disappearance in Finland journal (Muona et al., 2008, see Appendix has been explained as a consequence of the I) and were encouraged to search for the decline of its host-tree in managed forests. beetle throughout Finland. Finnish Considering that H. cruentatus has been government authorities were contacted and shown not to live on Populus in Finland at the species status was changed from all, this conclusion was incorrect and the “regionally extinct” to “endangered” on the conservation of aspens would not have Finnish Red List by the Ministry of helped the stabilization of this rare beetle Environment. The habitat of Hylochares in population. Vantaa was mapped and put under protection by the Southern Finnish The favoured habitat of Hylochares is rare Administration. Additionally, the general in Southern Finland and the value of the public was informed about these processes recently found site lies in its history. The through the largest Finnish newspaper current small rivulet has been a major river

“Helsingin Sanomat”. (Fig. 7, for In addition to the “local issue” how to most translation see Appendix II). effectively ensure the survival of H. cruentatus in Finland, a problem with The case of Hylochares is an example of wider implications has arisen: Seemingly the value of seemingly non-descript urban viable populations of several threatened wastelands. It also strongly accentuates the Finnish forest species are found in Russian need on leaving park-like habitats in urban Karelia right at the border to Finland. regions as natural as possible. Potentially Often their taxonomic status has not been important sites exist outside large tracts of assessed in detail and conservation unmanaged wilderness areas and clearing decisions in Finland are based on the shrubbery and dead wood or dense bushes biological observations of Russian because they look “untidy” are, from a populations. The conclusions about life- conservation point of view, inappropriate history and ecology drawn from this activities. Therefore, management practises knowledge may not always apply for their should rather mimic natural processes Finnish counterparts. In fact, the Finnish (Niemelä et al., 2007). populations might even be separate

species, as seen in the case of Hylochares.

It has to be emphasised that even if the

Russian and Finnish Hylochares populations would have been regarded as

conspecific due to their identical genetic

make-up, conservation actions based on

the knowledge obtained from the Russian

Hylochares would not have helped to save this rare beetle in Finland.

Conclusion

Mutation rates have become important molecular tools in fields such as

evolutionary and conservation biology. Yet

some of the methods relying on mutation

Fig. 7. Helsingin Sanomat, 09.12.2008 rates are regarded as controversial.

In this thesis two such methods, DNA lead to accurate divergence time estimates, barcoding of allied species and the strict at least for the invertebrates studied. metabolic clock were tested for their Although problems with these molecular accuracy and their ability to depict the tools exist, as shown, they by no means phylogenetic interrelationships and should be abandoned completely. For evolutionary history of the invertebrates DNA barcoding and the molecular clock, studied. Limitations of both methods were probably as many success stories found. (barcoding: e.g. Hebert et al., 2003; Hebert Using DNA barcoding the lack of power of et al., 2004; Clare et al., 2007; molecular mutations to distinguish between the clock: e.g. Renner 2005; Drummond et al., closely related Hylochares species was 2006; Rannala and Yang, 2007) as failures revealed. This shortcoming can be have been reported in the scientific explained due to the recent speciation literature (barcoding: e.g. Kaila and Ståhls, event of the beetles. Whereas mutations 2006; Meier et al., 2006; Wiemers and occur in a time course of million years, the Fiedler, 2007, molecular clock: e.g. Budd Finnish and Russian Hylochares and Jensen, 2004; Rodriguez-Trelles et al., populations probably have separated after 2004; Pulquério and Nichols, 2006). the last ice-age, only several thousands of It is crucial that DNA barcoding and years ago. Thus, not enough mutations molecular dating are applied with caution. have accumulated that could be detected in They at best are only simplistic methods order to differentiate the two taxa by that can never depict the complexity of sequencing the short barcoding region. biological systems. DNA barcoding should Also, the results of the molecular clock not be used instead, but along with study exposed drawbacks when mutations traditional taxonomy. Molecular clock were used for dating. It was shown here models need to be chosen carefully and that mutation rates of taxa in colder different clocks using biogeographical and climates can not be assumed a priori to be fossil calibration points should be tested. slower than rates of taxa in warmer Both methods, DNA barcoding and climates. Thus, correcting for temperature molecular dating, should embrace all data will not necessarily reconcile mutation and information available in order to rates in equally sized organisms. Hence, obtain comprehensive estimations of the applying a strict metabolic clock will not existing biodiversity as well as its evolutionary history.

Appendix I

Hylochares cruentatus (Gyllenhal) Suomessa – haapasepikästä halavasepikäksi (Col., Eucnemidae)

Jyrki Muona1, Noora Lassila2 & Lena Brüstle1

Muona, J., Lassila, N. & Brüstle, L. 2008: Hylochares cruentatus (Gyllenhal) Suomes- sa – haapasepikästä halavasepikäksi (Col., Eucnemidae) [Hylochares cruentatus (Gyl- lenhal) develops in willows, not aspen in Finland]. — Sahlbergia 14: 17–21. Helsinki, Finland, ISSN 1237–3273.

The biology and habitat requirements of the eucnemid Hylochares cruentatus (Gyl- lenhal) are described. It breeds in hard surface wood of broken trunks of Bay willow and Dark-leaved willow along regularly flooding small waters with untreated natural vegetation. Former records listing large aspen as the host tree refer to another, undescribed species not present in Finland. The biology is described in detail in the hope of obtaining further records of this rarity. 1Finnish Museum of Natural History, University of Helsinki, 2Undergraduate Library, University of Helsinki

Johdanto maastossa liikkuvat hyönteisharrastajat tämän avulla kiinnittäisivät huomiota uusiin esiinty- Hylochares cruentatus (Gyllenhal) on Euroopan misalueisiin ja ilmoittaisivat niistä tekijöille. puuhyönteisfaunan suurimpia harvinaisuuksia. Laji kuvattiin 1808 Suomesta, mutta löytöpaik- Elinympäristö ja lentoreiät koja kertyi vain tusinan verran seuraavien kah- den vuosisadan aikana (Muona 2008). Venäjän Tietomme lajin biologiasta perustuvat kolmen Karjalassa tehtyjen löytöjen perusteella lajin viime vuoden aikana tehtyihin kenttätöihin Van- uskottiin elävän erityisesti jättihaavoilla (Kan- taan kaupungin alueella. Halavasepikkää on ta- gas & Kangas 1944; Siitonen & Martikainen vattu kahdelta alueelta. Ne sijaitsevat muutaman 1994; Siitonen ym. 1996). Yllättävät uudet löy- kilometrin päässä toisistaan ja ovat epäilemättä döt Suomessa eivät tätä ajatusta tukeneet, isän- yhtä ja samaa esiintymää. Esiintymä on jakau- täpuiksi osoittautuivat halava (Salix pentandra) tunut kahdeksi pääalueeksi. Eteläiseltä alueelta ja mustuvapaju (Salix myrsinifolia) (Muona on löytynyt 17 runkoa, joista vain ennen vuot- 2008). Tarkempi tutkimus osoitti, että Suomen ta 2007 on kuoriutunut halavasepiköitä, ja 18 ulkopuolelta löytyneet yksilöt mitä ilmeisimmin runkoa, joista myös vuonna 2007 kuoriutui kuuluivat toiseen, kuvaamattoman lajiin (Muo- yksilöitä. Yhteensä sopivia puita on tutkittu yli na & Brustle, tekeillä). Uusien havaintojen 500. Pohjoisemmalta alueelta asuttuja runkoja perusteella lajin suomenkielinen nimi, haapa- tiedetään noin kymmenen ja entisiä syönnök- sepikkä, voidaan nyt siirtää itäisillä lähialueil- siä saman verran, mutta tältä alueelta löytynee la esiintyvälle lajille ja Hylochares cruentatus vielä useita uusia puita. Lentoreikiä ykkösalu- ristiä uudelleen. Isäntäkasvit ovat suosittu kanta eelta on laskettu yli 1200. Esiintymisalueet ovat sepikkänimille, joten napakka halavasepikkä on säännöllisesti tulviva jokiranta ja lintujärven valittu lajin uudeksi suomenkieliseksi nimeksi. kaltainen rehevä vesi sekä siitä laskeva pieni Kuvaamme lajin elinympäristön ja syön- joki. Runsaana esiintyvät mustuvapajut ja hala- nökset yksityiskohtaisesti siinä toivossa, että vat ovat olennainen osa elinympäristöä, koska 18 Muona ym.: Haapasepikästä halavasepikäksi

samoilla alueilla kasvavat raidat, istutetut pajut, lön koko elinkaari vaatii vain muutaman sentin lepät, haavat ja koivut eivät ole olleet asuttuja. puuta (kuva 3). Toukka syö käytävää jalokuori- Kaikki syönnökset ovat katkenneissa run- aismaisesti. Se tukee ruumiinsa ison etuselän ja goissa (kuva 1). Suurin osa rei’istä on alle met- eturinnan kitiinirakenteilla käytävän kattoon ja rin korkeudessa, mutta suurissa puissa niitä voi pohjaan ja jyrsii uutta käytävää päätään eri puo- olla laajalti ylempänäkin, korkeimmillaan niitä lille kääntämällä ja lopuksi työntää syönnöksen on havaittu viidessä metrissä. Reiät ovat usein tiukkaan pakettiin taakseen. Toukkanahkojen tiiviissä ryhmissä ja niitä voi olla satoja samal- vaihtopaikat löytyvät yleensä vanhojen pääkap- la alueella (kuva 2). Valtaosa reistä on kovas- seleiden avulla (kuva 3). Toukka on helposti sa kuorettomassa osassa puuta, jonka runko tunnistettavissa useimmista kuoriaistoukista ja- on yleensä muuten kuorellinen. Suurikokoisis- lattomuuden ja laajentuneen etuselän ja -rinnan sa mustuvapajuissa (korkeus 6–10 m) sepikät perusteella (kuva 6). Samankaltaisista jalokuo- voivat tulla suoraan kuoren läpi, mutta hala- riaisten toukista se eroaa etuselän kitiinilevyjen van kuoresta tätä ei ole havaittu. Reikien koko avulla, jalokuoriaisilla on vain yksi tällainen vaihtelee suuresti, läpimitta vaihtelee 1,9–4,2 rakenne. mm välillä (kuva 2). Tämä on paras ulkoinen Eteläisen Suomen tulvivat joet on usein tuntomerkki, koska minkään muun suomalaisen perattu ja niiden luonne on olennaisesti muut- hyönteisen kohdalla vaihtelu ei ole näin laajaa. tunut. Olemme tutkineet useita pääkaupunki- Jumien reikiä löytää samantyyppisistä puista, seudun ja Kirkkonummen näennäisesti sopivia mutta ne eivät ole läpimitaltaan yhtä vaihtele- rantoja löytämättä halavasepikkää. Laji kuvat- via ja suurimmatkin reiät ovat selvästi halava- tiin Paimionjoen rannalta Spurilan tilan alueelta sepikän suurten naaraiden lentoreikiä pienem- lahosta pajusta löydettyjen yksilöiden perus- piä. Aikuiset kuoriaiset ovat liikkeellä lähinnä teella. Levinneisyys on epäilemättä kattanut kesäkuun aikana (kuva 5). historiallisella ajalla eteläisen Suomen. Ainoal- la tällä hetkellä tunnetulla esiintymisalueella on Toukan ja aikuisen syönnös puussa poikkeuksellinen historia Vantaanjoen entisenä uomana, joka selittänee suurikokoisten paju- Yksittäisiä lentoreikiä ei pysty määrittämään kasvien määrän ja ihmisen vaikutuksen vähäi- tunkeutumatta puuainekseen. Puun sisällä syyden. Todennäköisesti vastaavia alueita ei ole syönnös on erittäin helppo tunnistaa. Toukkien useita löydettävissä, mutta erityisesti rehevien käytävät kulkevat kovassa pintapuussa ja ovat lintuvesien ympäristössä kannattaisi tätä harvi- hyvin leveitä ja litteitä ja niissä on paljon hie- naisuutta etsiä. noa, pakattua purua (kuva 4). Näiden käytävien lomassa ovat aikuisten täysin pyöreät ja tyh- Kirjallisuus jät kuoriutumiskäytävät (kuva 4). Samanlaista syönnöstä tekee Suomessa vain äärimmäisen Kangas, E., & Kangas, J. 1944: Über die Lebens- harvinainen kaulussepikkä, Melasis bupres- weise und die Larve von Xylophilus cruentatus Gyll. (Col., Eucnemidae). Suomen Hyönteistie- toides (F.), joka elää lähinnä lepillä ja tammel- teellinen Aikakauskirja 10 (1): 7-16. la. Sen lentoreiät ovat selvästi pienempiä ja sen Muona, J. 2008 Observations on the biology of Hy- toukkakäytävät kulkevat kohtisuoraan puun lochares cruentatus (Gyllenhal) (Col., Eucne-Eucne- syitä vasten, kun taas halavasepikän käytävät midae). Entomologica Fennica (painossa). mutkittelevat vapaasti. Siitonen, J. & Martikainen, P. 1994: Occurrence of rare and threatened insects living on decaying Halavasepikkä munii yksittäisiä munia joko Populus tremula: a comparison between Fin- vaurioituneeseen puun pintaan (Muona 2008) nish and Russian Karelia. Scandinavian Journal tai aikuisten ulostulokäytävien seiniin. Yksi- of Forest Research 9 (2): 185-191. Muona ym.: Haapasepikästä halavasepikäksi 19

Siitonen, J., Martikainen, P., Kaila, L., Mannerko- ski, I., Rassi, P., & Rutanen, I. 1996: New faunistic records of threatened saproxylic Coleoptera, Diptera, Heteroptera, Homop- tera and Lepidoptera from the Republic of Karelia, Russia. Entomologica Fennica 7: 69-76.

Kuva 1. Mustuvapaju (Salix �yrsinifolia), jonka katkenneessa haarassa on satoja halavasepikän (Hylochares cruentatus) lentoreikiä (kuva oikealla). Suurin osa toukista on kehittynyt haaran alaosassa puun kaakkoispuolella (kuva alla), �utta syönnös on levinnyt aina viiteen �etriin saakka. Kuvat Jyrki Muona 20 Muona ym.: Haapasepikästä halavasepikäksi

Kuva 2. Mustuvapaju (Salix myrsinifolia), jonka katkenneessa rungossa näkyy runsaasti erikokoisia halavasepi� kän (Hylochares cruentatus) lentoreikiä. Kuva Jyrki Muona

Kuva 3. Mustuvapajun (Salix myrsinifolia) kappale, jossa näkyy yksittäisen halavasepikän (Hylochares cruentatus) koko kehityskaari. � = �unintakohta vanhan aikuiskäytävän pohjalla. B = ensi��äisen toukkavaiheen pääkapseli. C = toisen toukkavaiheen pääkapseli. D = kol�annen toukkavaiheen pääkapseli. E = koteloitu�is� ka��io. F = aikuisen ulostuloaukko. Kuva Jyrki Muona Muona ym.: Haapasepikästä halavasepikäksi 21

Kuva 4. Halavasepikän (Hylochares cruentatus) syönnöksiä. Vase��alla �ustuvapajun (Salix myrsinifolia) �urtunut reuna, kapeita toukkakäytäviä ylhäällä, niiden alla pyöreä aikuisen käytävä. Oikealla yksityiskohta syönnöksestä halavalla (Salix pentandra), yläpuolella neljä toukkakäytävää, alla kaksi aikuisen ulostulokäytä� vää. Kuva Jyrki Muona

Kuva 5. Halavasepikkä naaras (Hylochares cruentatus), pi� tuus 7 ��. Kuva Jyrki Muona

Kuva 6. Halavasepikän toukka (Hylochares cruentatus), pi� tuus 10 ��. Kuva Jyrki Muona Appendix II abundance in Mätäoja, because the area was previously a bed of the Vantaa river. Mätäoja Valley will be protected in The wetland has also remained Vantaa exceptionally untouched, even though it is surrounded by housing areas. A rare beetle species inhabits a stream’s hollow The area that has been proposed for conservation is 27 hectares and the Mätäoja valley between Myyrmäki and majority of the land is owned by the city of Kaivoksela in Vantaa will be protected. Vantaa. Other valuable nature-areas are One of Europe’s most endangered beetles, found by Mätäoja. The marsh in its (halavasepikka), resides there. The beetle, northern part has been placed under formerly known as (haapasepikka) was protection because of the thought to have disappeared from Finland, sääskenvalkkukämmekkä flower, and the until it was re-discovered in Mätäoja in black alder wood that spreads along the 2004. The beetle requires bay willows and southern parts of Mätäoja. black willows in its habitat. These grow in

Acknowledgements Many thanks go to Jaakko Hyvönen and Ilari Sääksjärvi for kindly reviewing my First of all I would like to thank my thesis. supervisor Jyrki Muona for all his time, Tons of thanks to everyone involved in encouragement, stimulating scientific anyway in the Hotspots project, especially discussions and for teaching me how to thanks to the European Commission for answer my own questions. But also for my funding, Rosie Trevelyan and the reminding me that no matter what one Tropical Biology Association for the field should never forget one’s sense of humor. trips to Africa and Vincent Savolainen for

I am very grateful to Nicolas Salamin who making it all work. Special thanks to the provided me with so much help and advice other Hottiespotties, without you guys this and made it possible for me to stay at the PhD would not have been half as fun. Tia, University of Lausanne as long as I thanks for giving me a home in Lausanne needed, which turned out not to be 2 weeks and thanks to Cat for not letting me die of as planned, but 6 months. Merci to you and Malaria in Kenya. I wish you all the best everyone at Unil! for your future.

I would also like to thank Alfried Vogler at At the University of Helsinki I would like the British Museum of Natural History, to say how much I appreciate Juha Merilä Imperial College, for his input and and Leena Liikanen sorting out my suggestions regarding my work. finances and my contract. Many thanks go also to Veijo Kaitala and Ilkka Teräs for Further I would like to say a big thank-you answering my numerous questions and to Ward Wheeler who provided me with helping me with administrative issues. the opportunity to carry out laboratory Further thanks to everyone in the MES-lab, work for 4 months at such an amazing especially thanks to the “lunch crew”: place like the American Museum of Jaana, Leena, Maria and Minttu for Natural History in New York City. Thanks listening to all my crazy stories, your help also to all at the AMNH. and support in the lab and the funniest I also thank George Estabrook at Michigan lunches ever. Minttu, thanks for looking University who took the time to modified through bins to find my lost PCRs, for pink one of his computer programmes just so I sharks in pigeonholes, numerous chocolate could use it for my analyses. trips and for introducing me to Maija. Gunther, thanks not only for your help at

35 work but also for the long talks, the good To all the Prajogos, thanks for being my cooking, the best squash and for being “substitute family”, special thanks to Paula such a great friend. for all the fun! Kaisa, what can I say about someone who even knows my own social At the Finnish Museum of Natural History security number by heart? Thanks for I want to thank Gunilla Ståhls for being my housemate, personal assistant, providing me with data and for always cake supplier, entertainer, friend and much having an open door. Also thanks to all my more. friends and colleagues there. Special thanks to Diane, Jaska, Marianna, Misha I also want to say a big thank-you to my and Varpu. Varpu, you are for sure the family and close friends who are spread most amazing and interesting six-legged over all the corners of this globe. I cannot individual I have ever met. Marianna, mention you all here, but you know who thanks for your friendship and for always you are. being there for me. Thank you also for so Sinu, thanks for letting me into your world many other things that I will not even try to and for turning ordinary things, such as list them all here. escalator rides on rainy days, into magic Helsinki would not have been the same moments. without the people I have met outside my Last but definitely not least I would like to work. Special thanks to my former thank my parents. I can never thank you flatmates in Talonpojantie for giving me a enough for all the support and warm welcome in Finland. Criss, thanks encouragement you have given me. Your for being my first friend in Helsinki and belief in me gave me the strength to for our weekly dinner meetings. Of course believe in myself. This work I dedicate to thanks also to Imoh for joining us! you.

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