Secondary structure alignment and direct ... - Wiley Online Library

Secondary structure alignment and direct optimization of 28S rDNA sequences provide limited phylogenetic resolution in bark and ambrosia beetles (Curculionidae: Scolytinae) Blackwell Publishing Ltd

BJARTE JORDAL, JOSEPH J. GILLESPIE & ANTHONY I. COGNATO

Submitted: 17 May 2007 Accepted: 8 September 2007 doi:10.1111/j.1463-6409.2007.00306.x

Jordal, B., Gillespie, J. J. & Cognato, A. I. (2008). Secondary structure alignment and direct optimization of 28S rDNA sequences provide limited phylogenetic resolution in bark and ambrosia beetles (Curculionidae: Scolytinae). — Zoologica Scripta, 37, 43–56. Phylogenetic relationships in Scolytinae were reconstructed from 107 DNA sequences that spanned the D2 and D3 expansion segments, and related core regions of the nuclear large ribosomal subunit (28S). Sequences were analysed by parsimony and Bayesian analyses of aligned sequences aided by a new secondary structure model for the D2 –D3 domains. Direct optimization was performed on ambiguous alignment regions in combination with fixed states optimization of unambiguous regions, but performed poorly compared to the Bayesian and parsimony analyses. Generally, the phylogenetic signal mainly resolved relationships within tribes, while deeper divergences were either not resolved or received marginal support. In addition to confirming several previously established clades, we found that Micracini formed the sister group to Cactopinus, a group of mainly cactus feeding scolytine beetles. Furthermore, Ipini was monophyletic with Pseudips and Acanthotomicus subtending to the most basal node of that clade. The monophyly of Corthylini, which consists of the bark and cone feeding Pityophtorina and the ambrosia fungus-feeding Corthylina, was supported in some of the analyses. A close relationship was found between Phloeotribus and the two Phloeosinini genera Chramesus and Pseudochramesus, suggesting an evolutionary trajectory for the origin of a lamellate antennal club in Phloeotribus. Corresponding author: Bjarte Jordal, Department of Biology, University of Bergen, Allegt 41, NO-5007 Bergen, Norway. E-mail: [email protected] Joseph J. Gillespie, Department of Microbiology and Immunology, University of Maryland School of Medicine, 660 West Redwood Street, HH Room 3-24, Baltimore, MD 21201, USA Joseph J. Gillespie, Virginia Bioinformatics Institute, Bioinformatics Facility (0477), Washington Street, Virginia Tech, Blacksburg, VA 24061, USA. E-mail: [email protected]; [email protected] Anthony I. Cognato, Department of Entomology, Michigan State University, 243 Natural Science Bldg., East Lansing, MI 48824, USA. E-mail: [email protected]

Introduction Bark beetles in the weevil subfamily Scolytinae are well known for their severe effect on boreal forest stands and particularly so on the conifers. The economical impact from a handful of tree-killing species is huge and the commercial value of cut timber is often reduced by fungal pathogens transmitted by wood-boring ambrosia beetles (including the Platypodinae). What is less known to the broader audience, however, is the enormous variation in biological and ecological characteristics in the nearly 7500 species of Scolytinae and Platypodinae (Wood & Bright 1992). A variety of reproductive behaviours, ranging from monogyny to harem polygyny and inbreeding by sibling mating, are associated with different

genetic systems such as normal Xy diplodiploidy in outbreeding species, or haplodiploidy or paternal genome elimination for inbreeding species (see Jordal 2008). Resource use also goes far beyond the familiar association with inner bark and phloem, for example, there are many species from unrelated lineages that feed exclusively on fungal hyphae grown in wood tunnels (Beaver 1979). The non-reversed evolution of fungus cultivation (e.g. Farrell et al. 2001; Jordal 2002) has resulted in a tight symbiosis with highly modified cuticular modifications to carry fungal spores (Beaver 1989). The wide range of biological features across various groups of Scolytinae thus provides a highly informative system to study the evolution of important ecological traits. However,

© 2007 The Authors. Journal compilation © 2007 The Norwegian Academy of Science and Letters • Zoologica Scripta, 37, 1, January 2008, pp43–56

43

28S phylogeny of Scolytinae • B. Jordal et al.

detailed tests of key evolutionary innovations, for example those responsible for increased lineage diversification, are dependent on a robust phylogenetic hypothesis. In spite of an increased recent attention from evolutionary ecologists that use bark and ambrosia beetles as model systems in their research, a stable and predictive phylogeny is not yet in place. The classification of these beetles has changed frequently during the previous century and several recent phylogenetic hypotheses differ considerably between authors (see Kuschel et al. 2000; Jordal 2008; for further details). Wood (1973, 1978, 1982, 1993) proposed the first classification that claimed to be based on derived shared character states. Despite a rigorous treatment of morphological characters and their development, none of the proposed evolutionary relationships were tested in a cladistic or other phylogenetic framework. Thus, the first preliminary cladistic analysis of morphological characters for Scolytinae and Platypodinae was reported by Kuschel et al. (2000). Although as many as 80 morphological characters for 35 terminal taxa were included in that analysis, the nested position of Platypodinae within Scolytinae was only moderately supported, and relationships between scolytine taxa were largely unresolved. It was clear from Kuschel’s work that a broader taxon sampling and molecular data would help to test these hypotheses more rigorously. However, several recent molecular studies on higher level phylogeny of scolytine taxa have not been fully capable of resolving relationships between tribes and subfamilies (Normark et al. 1999; Sequeira et al. 2000, 2001; Farrell et al. 2001; Marvaldi et al. 2002). Two main complications may explain the lack of adequate resolution. First, taxon inclusion has either been undersampled in terms of numbers of taxa included or heavily biased towards boreal taxa. Second, the majority of sequenced genes exhibit non-optimal substitution rates unsuitable for resolving intertribal relationships (see, e.g. Sequeira et al. 2001: Fig. 2; Jordal 2007). This is particularly relevant to mitochondrial genes that generally evolve much faster than many nuclear genes in insects (Lin & Danforth 2004), although it should be noted that Cytochrome Oxidase I (COI) amino acids contain promising signal for ancient relationships within Scolytinae (Farrell et al. 2001; Sequeira et al. 2001; Jordal 2007). Among the nuclear genes, only Elongation Factor 1α (EF-1α) seems to have some potential for resolving relationships between tribes (Farrell et al. 2001). However, this gene alone is not sufficient for resolving deeper nodes. The conservative small subunit ribosomal RNA 18S should therefore be more appropriate but contains on the other hand very little phylogenetic signal within Scolytinae due to its low evolutionary rate (Farrell et al. 2001; Marvaldi et al. 2002). The large ribosomal subunit (28S) seems more promising in this respect as it contains several variable regions in addition to intervening conservative core rRNA regions. A recent analysis of partial 28S rRNA 44

sequences for conifer associated bark beetles provided some, but nevertheless limited phylogenetic signal in this group (Sequeira et al. 2000). We will therefore expand on this data set and explore more fully the phylogenetic potential based on secondary structure sequence alignments and direct optimization methods. Biological information is steadily increasing as an important component in the alignment of rRNA sequences (Morrison 2006). Instead of simply aligning sequences of variable lengths based on local profile matching and overall sequence similarity, the biological approaches utilize information from, for example, the secondary structure of the rRNA, or the evolutionary relationships between sequences. Because rRNA functions on the basis of its structure, several key features in the secondary structure are conserved between distantly related taxa (e.g. Kjer 1995; Gutell 1996). A predicted structure is obtained by comparing core helices and nonpairing regions from different sequences; the structural model thus provides an explicit and repeatable criterion for manual alignment (e.g. Gillespie et al. 2004). Another alignment procedure that incorporates biological information is direct optimization of sequences simultaneous with tree building (e.g. POY, see Gladstein & Wheeler 1997). Direct optimization combines statements on primary (alignment) and secondary (tree topology) homology, but it is not readily apparent from such analyses which sites may be homologous between sequences (e.g. Kjer 2004). An alternative is therefore to combine information from secondary structure with direct optimization, by constraining blocks of unambiguously aligned regions before optimization, in a procedure known as fixed-states optimization (Wheeler 1996). We present here a new sequence alignment for Scolytinae based on a new model for the D2 and D3 domains of 28S rRNA (see http://hymenoptera.tamu.edu/rna). The alignment includes sequences from nearly all scolytine tribes (23 of 26), and five genera of Platypodinae, to reduce potential artefacts from biased taxon sampling. New sequences were added from 14 genera in six tribes not previously included in any 28S phylogenies, in addition to 12 more genera from tribes previously included (see Table 1). With the addition of new taxa, we aim to test the monophyly of most Scolytinae tribes as currently classified (Wood 1986) and to test several unsettled relations between higher taxa. Some of these hypotheses includes: (i) Ctenophorini is basal in Scolytinae and closely related to Scolytini (Wood 1986); (ii) the monotypic Cactopinini is related to the Nearctic Micracini; (iii) Crypturgini is closely related to Dryocoetini; (iv) Dryocoetini includes Xyleborina and forms the sister group to Ipini; and (v) Premnobius forms the sister lineage to Ipini. To enable comparison of the phylogenetic utility at different hierarchical levels, we have furthermore included multiple species from five genera of Ipini.

Zoologica Scripta, 37, 1, January 2008, pp43–56 • © 2007 The Authors. Journal compilation © 2007 The Norwegian Academy of Science and Letters

B. Jordal et al. • 28S phylogeny of Scolytinae

Table 1 Taxa and GenBank number. No.

Genus/species

Subfamily

Subtribe

Genbank no.

No.

Genus/species

Subfamily

Subtribe

Genbank no.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54

Araucarius major Araucarius minor Stenancylus sp. Platypus incompertus Chaetastus montanus Crossatarsus wallacei Dinoplatypus pseudocupulatus Euplatypus parallelus Cnesinus lecontei Cnesinus sp. Cactopinus rhois Araptus sp. Conophthorus ponderosae Corthylus sp. Dendroterus striatus Monathrum dentigerum Monathrum mali Pseudopityophthorus sp. Cryphalus kesiyae Hypothenemus setosus Hypothenemus sp. Crypturgus alutaceus Dolurgus pumilus Gymnochilus reitteri Pycnarthrum hispidum Scolytodes acuminatus Diamerus curvifer Strombophorus celtis Coccotrypes dactyliperda Xylocleptes bispinus Hylastes porculus Hylurgops rugipennis Alniphagus aspericollis Hylesinopsis dubius Hapalogenius seriatus Hylesinus varius Sueus niisimai Chaetophloeus sp. Hypoborus ficus Liparthrum nigrescens Acanthotomicus kepongi Ips acuminatus Ips amitinus Ips avulsus Ips bonanseai Ips calligraphus Ips cembrae Ips emarginatus Ips grandicollis Ips hoppingi Ips knausi Ips lecontei Ips pilifrons Ips plastographus

Cossoninae Cossoninae Cossoninae Platypodinae Platypodinae Platypodinae Platypodinae Platypodinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae

Araucariini Araucariini Rhyncolini Platypodini Tesserocerini Platypodini Platypodini Platypodini Bothrosternini Bothrosternini Cactopinini Corthylini Corthylini Corthylini Corthylini Corthylini Corthylini Corthylini Cryphalini Cryphalini Cryphalini Crypturgini Crypturgini Ctenophorini Ctenophorini Ctenophorini Diamerini Diamerini Dryocoetini Dryocoetini Hylastini Hylastini Hylesinini Hylesinini Hylesinini Hylesinini Hyorrhynchini Hypoborini Hypoborini Hypoborini Ipini Ipini Ipini Ipini Ipini Ipini Ipini Ipini Ipini Ipini Ipini Ipini Ipini Ipini

AF308350 AF308351 AF375301 AF375298 AF375299 EU090336 AF375302 AF375304 AF308352 AF308353 EU090343 AF375297 EU090330 EU090344 EU090339 EU090346 EU090331 AF375305 EU090338 AF308389 AF308388 EU090328 EU090355 EU090353 EU090352 EU090351 EU090348 AF308355 AF375300 EU090347 AF308387 AF308364 AF308367 AF308356 AF308368 AF308365 AF308354 AF308369 EU090350 AF308370 EU090306 EU090296 EU090315 EU090297 EU090313 EU090319 EU090320 EU090325 EU090323 EU090321 EU090327 EU090324 EU090311 EU090309

55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107

Ips sexdentatus Ips tridens Ips typographus Ips woodi Orthotomicus caelatus Orthotomicus erosus Orthotomicus latidens Orthotomicus longicollis Orthotomicus mannsfeldi Orthotomicus suturalis Pityogenes bistridentatus Pityogenes hopkinsi Pityogenes irkutensis Pityogenes porifrons Pityokteines elegans Pityokteines ornatus Pseudips concinnus Pseudips mexicanus Hylocurus hirtellus Micracis carinulatus Chramesus asperatus Chramesus sp. Phloeoditica sp. Phloeosinus punctatus Pseudochramesus sp. Phloeotribus liminaris Phloeotribus puberulus Phrixosoma sp. Scolytus unispinosus Scolytus ventralis Scolytoplatypus entomoides Scolytoplatypus sp. Dendroctonus mexicanus Dendroctonus murrayanae Dendroctonus ponderosae Dendroctonus pseudotsugae Dendroctonus terebrans Hylurgonotus antipodus Hylurgonotus tuberculatus Pseudohylesinus sp. Pseudohylesinus nebulosus Sinophloeus porteri Xylechinosomus valdivianus Amasa schlichi Ambrosiodmus sp. Euwallacea piceus Anisandrus hirtus Premnobius cavipennis Premnobius sexdentatus Ctonoxylon flavescens Trypodendron lineatum Xyloterinus politus Xyloterinus politus

Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae Scolytinae

Ipini Ipini Ipini Ipini Ipini Ipini Ipini Ipini Ipini Ipini Ipini Ipini Ipini Ipini Ipini Ipini Ipini Ipini Micracini Micracini Phloeosinini Phloeosinini Phloeosinini Phloeosinini Phloeosinini Phoeotribini Phoeotribini Phrixosomini Scolytini Scolytini Scolytoplatypodini Scolytoplatypodini Tomicini Tomicini Tomicini Tomicini Tomicini Tomicini Tomicini Tomicini Tomicini Tomicini Tomicini Xyleborina Xyleborina Xyleborina Xyleborina Premnobiini Premnobiini Xyloctonini Xyloterini Xyloterini Xyloterini

EU090314 EU090310 EU090298 EU090322 EU090317 EU090302 EU090300 EU090301 EU090312 EU090308 EU090304 EU090303 EU090305 EU090307 EU090318 EU090326 EU090316 EU090299 EU090332 AF375303 AF308362 AF308359 AF308358 AF308361 AF308360 AF308373 AF308372 EU090349 AF375306 EU090342 EU090345 AF308391 AF308383 AF308384 AF308385 AF308374 AF308386 AF308376 AF308375 AF308378 AF308379 AF308377 AF308366 EU090329 EU090337 EU090340 EU090335 EU090341 EU090354 AF308392 AF308394 AF308395 EU090334


45


Materials and methods Taxa examined and sequencing We augmented 49 published sequences (Sequeira et al. 2000; Farrell et al. 2001) with the inclusion of 58 new sequences as listed in Table 1. DNA was extracted following protocols of Cognato & Vogler (2001) or by the Qiagen DNEasy kit; the remaining body parts were pinned and vouchered at the A.J. Cook Arthropod Research Collection, Michigan State University, Harvard Museum of Comparative Zoology, or in the research collection of BHJ. The D2 and D3 of 28S DNA were amplified with the following general beetle primers: D2 UP-4 (forward) 5′-GAG TTC AAG AGT ACG TGA AAC CG-3′, D2UP-COL1 (forward) 5′-CCG TTG AGG GGT AAA CCT GAG AAA C-3′, D2 DN-B (reverse) 5′-CCT TGG TCC GTG TTT CAA GAC-3′ and 28S-B (DN) (reverse) 5′-TCG GAR GGA ACC AGC TAC TA-3′ (Gillespie et al. 2004). The following scolytine specific 28S D2 primer was also developed: 28Sscol1 (forward) 5′-AAC GAA AGG TCG AAG GAA G-3′. Primers used for the 28S-D3 are from Whiting et al. (1997). Each PCR reaction contained: 35 µL ddH2O, 5 µL 10x TaqDNA polymerase buffer (Promega, Madison, WI), 4 µL 25 mM Promega MgCl2, 1 µL 40 mM deoxynucleotide triphosphates (dNTPs), 2 µL of each Promega TaqDNA polymerase and 1 µL of DNA template. PCR was performed on a thermal cycler (MJ Research, Waltham, MA) under the following conditions: one cycle for 2 min at 95 °C, 34 cycles for 1 min at 95 °C, 0.75 min at 50 °C, 1 min at 72 °C and a final elongation cycle of 5 min at 72 °C. Purified PCR products were directly sequenced using BigDye® Terminator v.1.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA). Edited sequences were deposited in GenBank (Table 1). Multiple sequence alignment All sequences were aligned manually according to secondary structure (Kjer 1995), with notation following Gillespie et al. (2004). Alignment initially followed the secondary structural model of Chrysomelidae 28S rRNA D2 and D3 expansion segments and related core rRNA regions (Gillespie et al. 2004). Regions unique to scolytid beetles were newly annotated, and thus, slightly adjusted according to the Chrysomelidae model. All regions variable in sequence length and base composition, especially hairpin-stem loops and large insertions not present in the chrysomelid model, were evaluated in the program mfold (version 3.1; http:// bioinfo.math.rpi.edu/~zukerm/), which folds RNA sequences based on free energy minimizations (Mathews et al. 1999; Zuker 2003). These free energy-based predictions were used to facilitate the search for potential base-pairing helices, which were confirmed only by the presence of compensatory base changes across a majority of taxa. The alignment was annotated with a pairing mask equivalent to that utilized in 46

the program PHASE ( Jow et al. 2002; Hudelot et al. 2003). The mask is a parenthetical/dot statement that distinguishes between pairing and non-pairing regions of the sequences. Regions in which positional homology assignments were ambiguous across all taxa were defined according to structural criteria as in Kjer (1997) and described as regions of alignment ambiguity (RAA), regions of slipped-strand compensation (RSC), or regions of expansion and contraction (REC) following the methodology of Gillespie (2004). Ambiguously aligned regions were enclosed within brackets. A text file of the complete multiple sequence alignment is posted at AIC’s (http://www.hisl.ent.msu.edu) and the jRNA (http://hymenoptera.tamu.edu/rna) websites with specific explanations regarding the rRNA structural alignment. Scripted manipulation The jRNA website contains perl scripts (Jrna scripts) for the processing of structurally aligned data sets (Matt Yoder, Ohio State University). In short, the Jrna scripts are designed to integrate information from three files: (i) A structural alignment annotated according to Gillespie et al. (2004) that is also an executable NEXUS file. (ii) A helix index file that identifies each white-space delimited block and each stem-pair in the alignment; and (iii) A perl script that is specific for the data set under investigation. The Jrna scripts were used to parse our alignment and helix index file (with pairing statement) and return input file formats for several phylogenetic programs (see below). Where applicable the scripts integrated the pairing mask and information pertaining to helix, non-helix and non-homologous regions into input parameters for subsequent analysis. These features, as well as all input files generated in this study, are available at the jRNA website following the links to Scolytinae model. Phylogenetic analysis Parsimony and likelihood analyses were both performed on the same structurally aligned character matrix, with ambiguously aligned regions removed (see above). Thus, 1057 characters were included in these analyses. For the parsimony analysis, most-parsimonious reconstructions were obtained by heuristic searches with 500 random stepwise addition replicates using PAUP* (Swofford 2002). Gaps were treated as missing data or as a fifth character state. Bootstrap values were determined by performing 500 pseudo-replicates of 20 random addition replicates each. Because the majority of taxa included missing data at the 3′ end, we also performed similar analyses on a shorter matrix of 772 characters. We also analysed the data under maximum likelihood using Bayesian estimation of phylogeny as implemented in PHASE 2.0 (Jow et al. 2005). PHASE allows for mixed model analysis, in which both pairing and non-pairing regions can be modelled under likelihood and integrated into the analysis



simultaneously. In PHASE 2.0 multiple models for pairing regions are available ( Jow et al. 2005); however, several studies have suggested that seven-state models for RNA basepairs are likely better than six- and 16-state models (Savill et al. 2001; Gillespie 2005). This is because seven-state models include parameters for non-canonical basepairs (unlike six-state models), yet lump these infrequent substitutions into one class (unlike the 16-state models that parameterize all types of non-canonical basepairs). While several studies using PHASE have utilized the most general reversible sevenstate model (model 7A) ( Jow et al. 2002; Hudelot et al. 2003; Gillespie et al. 2005), it has recently been suggested that a simpler seven-state model, which still assumes non-zero rates of double substitutions but does not allow for basepair reversal asymmetry, is likely a better model with far fewer parameters than model 7A (Gibson et al. 2005). This model (model 7D) was incorporated in all of the PHASE analyses. Results from MODEL T EST (Posada & Crandall 1998) suggested modelling non-pairing regions of the alignment under the most general time reversible model with discrete gamma categories and proportion of invariant sites included (GTR + Γ + I). Flat priors were used for all analyses. Four Markov chains were used in an effort to decrease time until convergence (Ronquist & Huelsenbeck 2003). Three runs of the same analysis starting from randomly different sampling spaces (seeds) were used. Ten million generations were run to ensure that sampling adequately explored the parameter space. The degree of convergence in tree topologies, clade posterior probabilities and parameter posterior probabilities across all analyses were analysed in the program TRACER ver 1.2 (Rambaut & Drummond 2004), which provides graphical plots and numeric reports of the estimated sample size (ESS). Length variable regions are often very informative for phylogeny estimation (reviewed in Lee 2001), especially in local levels of estimated trees. In an effort to evaluate the relative signal within ambiguously aligned regions, we performed direct optimization (Sankoff et al. 1973; Sankoff 1975; Kruskal 1983; Sankoff & Cedergren 1983) on the alignment using the program POY ver. 3.0.11 (Gladstein & Wheeler 1997). We used our structural alignment to objectively divide the data into 218 blocks, or input files, 26 of which were excluded due to zero information content within them (single autapomorphies). Eighty-three blocks were designated as ‘prealigned’, meaning that no insertions or deletions occurred within them across all taxa. We used the optimization alignment (OA) approach of Wheeler (1996) on all blocks, initially setting the gap–transition–transversion matrix (ts : tv) to 1 : 1 : 1. An additional analysis was performed with the gap : ts : tv matrix set 2 : 1 : 1. The following command line was used to complete our POY analyses: -noprealigned data/ pd000 -prealigned data/pd001 -noprealigned data/pd002

-prealigned ...*... -prealigned data/pd216 -prealigned data/pd217 -prealigned data/pd218 -terminalsfile data/poybatch_terminals -seed 865 -minterminals 0 -checkslop 10 topooutgroup AF308351_Ara_mino_COSSO -replicates 20 -nooneasis -buildsperreplicate 20 -tbr -spr -maxtrees 10 -drifttbr -numdrifttbr 1 -treefusespr -treefusetbr -fusingrounds 1 -ratchettbr 1 -molecularmatrix g1tv1ts1.txt -characterweights -printtree -plotwidth 100 -poytreefile scol.poytreefile.txt -poybintreefile poybatch.poybintree.txt -plotechocommandline -phastwincladfile winclad.out. Note: the asterisk in the command line above denotes where the majority of the input file names were removed for brevity. The full command line is available at the following websites (http:// www.hisl.ent.msu.edu and http://hymenoptera.tamu.edu/rna). To compare the performance of POY, we also included the length variable regions in a PAUP parsimony analysis. The same tree search conditions, as described above, were used for 1640 characters. The length variable regions were aligned by comparison of related OTUs (i.e. those that occur in the same tribe) so to increase the likelihood of assessing positional homology. Patterns of nucleotides occurred in these regions and many obvious alignments were apparent (see text file at the above mentioned websites). Alignments of other nucleotides in these regions were questionable and the inclusion of some homoplastic comparisons likely occurred.

Results Parsimony analysis The parsimony analyses produced rather different topologies, depending especially on the coding of gaps (fifth character or missing). Only when gaps were treated as a fifth character, did Pityogenes and Ips form well supported monophyletic sister groups. On the contrary, Corthylini (ex Dendroterus) was only monophyletic when gaps were treated as missing. Matrices with gaps coded as a fifth character generally produced the highest number of bootstrap supported nodes (Table 2). Whether or not the extra 285 characters in the 3′ end were included (resulting in missing data for 87 of the 107 taxa) had little influence on the results; the number of resolved and bootstrap supported nodes and their support values remained essentially the same. Topological congruence across all parsimony analyses included monophyly of Hylastini, Micracini, Ipini, Xyloterini and Platypodini. Hylastini was sister to Dendroctonus and nested within a highly paraphyletic Tomicini. Only two of the three Hypoborini taxa, Liparthrum and Hypoborus, grouped together. Phloeotribus was nested within a clade consisting of Chramesus and Pseudochramesus, but not other Phloeosinini. Micracini grouped consistently with the single included species of Cactopinus. Xyleborina formed a well supported clade that included Coccotrypes, however, without Premnobius. The latter taxon instead grouped with low support to Phrixosoma. Within the more densely sampled Ipini we usually


47


Table 2 Support for higher clades under different types of analyses. The first six columns correspond to parsimony analyses: the first two columns indicate the support obtained for the main data set (1057 characters) where ambiguous sites have been excluded; columns 3 and 4 (772 characters) with 285 characters deleted from the 3′ end; columns 5 and 6 (all characters) with all arbitrarily aligned indel regions included. Bootstrap analysis was not possible for the POY analyses and ‘*’ indicate nodes present in the strict consensus tree. A hyphen indicates nodes that were not present in a specific analysis; when included in parentheses, at least some trees contradicted the specified node. Tree search parameters Clade

Gap = ?

Gap = 5th

772 char, gap = ?

772 char, gap = 5th

All char, gap = ?

All char, gap = 5th

Dendroctonus Dendroctonus + Hylastini Hylastini Tomicini in Araucaria Alniphagus + Phloeoditica Phloeosinini (part) + Phloeotribus Liparthrum + Hypoborus Micracini Micracini + Cactopinini Ips Pityogenes Pityogenes + Ips Pseudips + Acanthotomicus Ipini Xyleborina + Coccotrypes Xyloterini Corthylini, ex Dendroterus Corthylini Platypodini Nodes w/BP > 50 % Nodes w/BP > 90 %

99 < 50 64 – < 50 < 50 93 78 < 50 – – – < 50 < 50 98 98 93 – 99 32 19

100 < 50 68 – < 50 < 50 99 86 < 50 67 100 55 < 50 < 50 100 100 – – 98 45 22

100 < 50 65 – < 50 < 50 92 86 < 50 – – – < 50 < 50 99 98 92 – 100 35 22

98 < 50 65 – < 50 < 50 95 77 < 50 61 93 < 50 < 50 < 50 100 99 – – 98 42 21

100 – 92 – – 63 93 80 63 86 100 (–) 64 67 98 96 93 < 50 99 44 23

100 < 50 70 – – 91 98 50 91 66 97 62 92 85 82 97 – – 100 45 25

recovered monophyletic genera of Ips, Pityogenes and Pseudips. The two species included from the latter genus grouped with Acanthotomicus as the most basal clade in Ipini (Figs 1 and 2). Bayesian analysis The clade containing Dendroctonus and Hylastini was one of very few groups found by the parsimony analysis that was not supported in the Bayesian analysis (Fig. 3). Instead, Hylastini was replaced by Pseudohylesinus. The Bayesian analysis furthermore supported all of Ipini subclades that were supported in the parsimony analyses based on gaps as a fifth character. In addition, the Bayesian topology also showed a monophyletic Corthylini and a group of tomicine taxa that are all associated with Araucaria trees (Sinophloeus, Hylurgonotus, Xylechinosomus). Posterior probabilities were generally higher than the bootstrap values obtained in the parsimony analysis. Analyses of full length sequences The topology resulting from direct optimization of all nucleotides in POY contained very little resolution among higher clades (Fig. 4). Only 10 of the 19 clades listed in Table 2 were recovered in this analysis. However, Ipini, Xyleborina + Coccotrypes, Xyloterini, Platypodini and Micracini + Cactopinus 48

1:1:1

Bayesian (Phase)

* – * – – * – – * – * – * * * * – – * na na

100 – 100 77 90 97 86 78 97 63 100 69 74 81 100 100 86 70 100 69 41

POY

were all monophyletic. Some other interesting clades from the other analyses were also recovered in the POY analysis, such as the Phloeotribus–Pseudochramesus–Chramesus clade. On the other hand, Hypoborus did not group with the closely related taxon Liparthrum, Corthylini was paraphyletic with respect to the cossonine Stenancylus, and Dendroctonus grouped with a conglomerate of putatively highly unrelated taxa. Increased gap cost (2 : 1 : 1) resulted in nearly the same topology. To further evaluate the usefulness of length variable regions in the phylogenetic analysis, we also conducted a parsimony analysis which included all sites (1640 characters). Intuitively, data from length variable regions will likely provide more phylogenetic information for recent divergences (where indel regions are of same length) than more ancient divergences. Nevertheless, inclusion of length variable regions resulted in similar or slightly higher bootstrap supported nodes compared to the analyses of unambiguously aligned nucleotides. In fact, 8 of the 20 higher clades listed in Table 2 received considerably higher bootstrap values than in the more restricted analyses of unambiguously aligned regions. Several of the deeper divergences also increased their bootstrap support, for example Hylastini, Ipini and the Micracini–Cactopinini clade, and furthermore resulted in a monophyletic Corthylini.



Fig. 1 Strict consensus of 60 most parsimonious trees resulting from the analysis of 1057 unambiguously aligned characters, gaps treated as a fifth character (L = 3720, 378 parsimony informative characters, CI = 0.30, RI = 0.53). Bootstrap support values higher than 50% are shown above the nodes. The topology was rooted with two species of Araucarius and one Stenancylus.


49


Fig. 2 One of the 60 most parsimonious trees, with branch lengths (see Fig. 1 for further details).

50



Fig. 3 A 50% majority-rule consensus tree of the 28 000 trees sampled in the Bayesian analysis of 28S data. Pairing and non-pairing regions were modelled separately. A seven-state model was implemented for the paring regions and a most general time reversible model with discrete gamma categories and proportion of invariant sites included was used for the non-pairing regions. See text for details of the analysis. Numbers above branches indicate posterior probabilities.


51


Fig. 4 Majority rule consensus tree of 360 most parsimonious trees resulting from direct optimization in POY. Gaps, transitions and transversions received the same weight (1 : 1 : 1). Numbers above the nodes show the proportion of trees that fits this topology. See Materials and methods section for details.

52



Discussion Utility of 28S rDNA sequences in Scolytinae phylogeny The compiled set of 28S sequences demonstrated only limited potential in resolving relationships between tribes and higher level of Scolytinae. Less than half of the 108 possible nodes obtained bootstrap values above 50% in any parsimony analysis and a large number of basal nodes were unresolved in the POY and Bayesian topologies. A somewhat stronger signal in more derived clades was apparent from the Bayesian analysis, resulting in posterior probabilities higher than 50% in more than 65% of the nodes. However, posterior probabilities are generally inflated compared to other kinds of node support (Cummings et al. 2003) and thus do not divert from our main conclusion that the phylogenetic signal is not very robust for the most basal portion of the tree topology. Two main factors tend to be responsible for low resolution in phylogenetic analyses — either biased taxon sampling or unsuitable substitution rates. Because taxon sampling in this study was well balanced and much improved from previous phylogenetic studies, it seems unlikely that common phenomena associated with undersampling (such as soft polytomies and long branch attraction) should be responsible for the ephemeral assemblages of largely unsupported basal nodes. Thus, it seems more likely that the evolutionary rate of the sequenced regions of 28S is unsuitable for resolving deep divergences within Scolytinae. Consistent with our suggestion that the substitution rate might be too high for resolving deeper divergences, we found largely resolved nodes in more recent branches of the phylogenetic tree, such as many intratribal relationships (especially Ipini, Micracini, Xyleborina and Corthylini). However, substitution rates varied tremendously between clades (inferred from branch length differences) without necessarily being negatively correlated with node support. For instance, the strongly supported Platypodini clade had rates more than double of the rate in Ipini and tree times the rate for Xyleborina, yet they all revealed similarly strong node support (cf. Figs 1 and 2). High substitution rate is therefore a far too simplified explanation for the lack of resolution, especially because exclusion of indel rich regions did not improve the analyses. Rather, the addition of the most variable regions of the D2 and D3 domains increased the support for many nodes in the parsimony analysis of all characters. Alignment of length variable regions is conceivably the most critical aspect in successful tree reconstruction from ribosomal gene sequences. We believe that our approach that involved secondary structure provides the most reliable method by assuring high levels of homology through the confirmation of compensatory base changes across the majority of taxa. This approach may nevertheless appear overly conservative, given that 35% of the characters were excluded and may limited the resolution by including a prohibitively low number of informative characters (PI = 378).

Some proponents of secondary structure alignments have therefore suggested that ambiguous regions may be analysed further by direct optimization, fixing the prealigned unambiguous regions before optimization (e.g. Gillespie et al. 2005; Kjer et al. 2007). Our application of fixed states optimization nevertheless produced limited resolution, with relatively few of the well supported groups suggested by the Bayesian and parsimony analyses recovered in the POY analyses. The poor performance by direct optimization was contrasted by the positive contribution to node support in the parsimony analyses when all ambiguous sites were included. Even though these characters were quite crudely aligned, the signal provided by a low proportion of homologous characters more than outweighed the higher proportion of homoplastic characters. It is clear that alignment of ambiguous regions introduces lower confidence in primary homology statements and thus become controversial from a philosophical perspective (e.g. Kjer et al. 2007). However, we have shown in this study that more information can potentially be extracted from ambiguous alignment regions and these alignments could be further improved by multiple alignment software. Taxonomic implications Despite the generally low resolution in tree topologies, our results agreed on several interesting phylogenetic relationships and supported many previously established clades. The enigmatic Cactopinini was included in a phylogenetic analysis for the first time and came out as sister to the Micracini in all analyses. Only two of the analyses provided high node support for this group, but we note that preliminary analyses of EF-1α also support this relationship (BHJ, unpublished data). A sister relationship between the two tribes is also expected from morphological similarity ( Wood 1986), for example several characteristics of the pronotum and elytra, but especially the narrow and obliquely truncated protibiae which have few socketed teeth along the apical margin. The 19 species of Cactopinus are mainly found in the Mexican plateau region ( Wood & Bright 1992), at the centre of diversity for the Neotropical Micracini. Both groups share a similar preference for dry forest types where they often breed in dry woody tissue. This study is also the first to analyse phylogenetically a broad sample of Corthylini, including several species each of the fungus cultivating Corthylina and the bark and cone feeding Pityophtorina (Wood 1982, 1986). Corthylini was monophyletic in the Bayesian and one parsimony analysis, while all except Dendroterus were monophyletic in two additional parsimony analyses (all with gaps coded as missing data). We therefore conclude that Corthylini is most likely monophyletic. Among the best resolved and supported groups was the Ipini, in which the genera Ips, Pityogenes and Pseudips were monophyletic in most analyses. The latter genus was clearly


53


distinct and confirmed the recent separation from Ips (Cognato 2000). The placement of Orthotomicus longicollis in Pityokteines seems odd at first, but preliminary data from EF-1α actually support the placement inside, or close to, Pityokteines. In general, the relationship of Ipini species suggested by the Bayesian and parsimony topologies was nearly the same as those found in Cognato & Vogler (2001), especially within Ips. Among the several confirmed results from previous studies we note that Hylastini was nested within a paraphyletic Tomicini (as in Sequeira et al. 2001), usually as the sister to Dendroctonus (as in Sequeira et al. 2000; Farrell et al. 2001). It is also interesting to note that the Bayesian analysis resulted in the monophyly for the four Neotropical species associated with Araucaria. These species have been hypothesized to be rather basal in the first wave of conifer associations in the late Cretaceous (Sequeira et al. 2000). Their placement relative to other Scolytinae was unfortunately not possible to estimate with the current data set. We also confirmed a close relationship between Phloeotribus and Pseudochramesus–Chramesus, which may indicate the evolutionary pathway for the unique origin of the lamellate antennal club in Phloeotribus. Species of the Pseudochramesus have a strongly asymmetric club with diagonal sutures quite similar to some Phloeotribus when they keep the three lamellae of the club together. In accordance with recent molecular studies (see Normark et al. 1999; Farrell et al. 2001; Jordal 2002; Jordal et al. 2002) we found a close relationship between the inbreeding and haplodiploid Coccotrypes (Dryocoetini) and the Xyleborina. Some previous classifications of Xyleborina have included Premnobius as the most basal taxon in that group (see, e.g. Wood & Bright 1992); however, recent molecular studies support a closer relationship to the Ipini for this genus (see Normark et al. 1999; Farrell et al. 2001; Jordal et al. 2002). Although we could not confirm the affiliation to Ipini in this study, the genus was not closely related to Xyleborina. Species of Premnobius are morphologically rather different from xyleborines, and deviate strongly in the shape of the labial palpi and the proventriculus in adults, and in the spiracles of the larvae (Browne 1961; Jordal 2001). They are also biologically unique by constructing distinct pupal cradles before metamorphosis. We therefore support Browne (1961) classification of Premnobius in a separate tribe, Premnobiini, as recently supported by Bright & Torres (2006). Several hypothetical relationships predicted from previous morphological studies (e.g. Wood 1978) were not supported in this study. However, the paraphyly of Hylesinini and Tomicini, Hypoborini, Diamerini, Phloeosinini and Cryphalini was also reported by Farrell et al. (2001), suggesting that tribe monophyly might not be a viable hypothesis in future analyses. Several other tribes were also paraphyletic, albeit more unexpectedly as these groups were previously support by either a large amount of morphological data or by molecular 54

data. The Ctenophorini is a rather homogeneous group morphologically (Wood 1978; Jordal 1998) and the complete lack of phylogenetic signal to resolve this tribe was surprising. Dolurgus and the other genera of Crypturgini are, despite the 28S rRNA results here, a strongly supported monophyletic group based on morphological and four molecular data sets ( Jordal & Hewitt 2004). Premnobius did not group together with Ipini as the sister clade to Dryocoetini and Xyleborina as has been well documented in several recent reports (Normark et al. 1999; Farrell et al. 2001; Jordal et al. 2002). Finally, Platypodini was monophyletic, but did not group with the tesserocerine Chaetastus. The sequence of C. montanus was much shorter than other sequences, however, and it might be that the larger number of missing data in this sequence resulted in the less confident placement of this taxon. Platypodinae is otherwise a very strongly supported group of beetles (Kuschel et al. 2000; Farrell et al. 2001; Marvaldi et al. 2002). Future directions We have in this study presented a phylogenetic analysis of 28S rDNA sequences that were aligned according to a new secondary structure model of the expansion segments D2–D3 and related core rRNA. The most conservative inclusion of unambiguous alignment regions did not provide much resolution beyond previous knowledge about Scolytinae phylogeny. It nevertheless seems far fetched to exclude 28S rDNA data in future phylogenetic analyses of the group, especially with regard to the sometimes pronounced hidden support from ribosomal genes in combined analyses for deeper nodes (see, e.g. Cognato & Vogler 2001; Damgaard & Cognato 2003). Alignment based on secondary structure is very labour intensive, however, and the limited resolution obtained from this gene fragment may suggest that protein encoding genes offer the greatest promise for resolving a Scolytinae phylogeny. Recently established primers and protocols, for example EF-1α (Normark et al. 1999), CAD (rudimentary) and arginine kinase will likely augment rDNA-based phylogenies in the future (Jordal 2007) and allow for a more complete assessment of the inherent phylogenetic signal within rDNA sequence data.

Acknowledgements We thank Claire McKenna for help with the generation of sequence data and Krishna Dole for help with POY analyses. This work was partially funded by a NSF-PEET grant (DEB-0328920) to AIC. BHJ was funded by grant no. 170565/V40 from the Norwegian Research Council.

References Beaver, R. A. (1979). Host specificity of temperate and tropical animals. Nature, 281, 139 –141.



Beaver, R. A. (1989). Insect–fungus relationships in the bark and ambrosia beetles. In N. Wilding, N. M. Collins, P. M. Hammond & J. F. Webber (Eds) Insect–Fungus Interactions (pp. 121–143). London: Academic Press. Bright, D. E. & Torres, J. A. (2006). Studies on West Indian Scolytidae (Coleoptera). 4. A review of the Scolytidae of Puerto Rico, USA, with descriptions of one new genus, fourteen new species and notes on new synonymy. Koleopterologische Rundschau, 76, 389 –428. Browne, F. G. (1961). The generic characters, habits and taxonomic status of Premnobius Eichhoff (Coleoptera, Scolytidae). Fourth West African Timber Borer Research Unit Report, 1961, 45 – 51. Cognato, A. I. (2000). Phylogenetic analysis reveals new genus of Ipini bark beetle (Scolytidae). Annals of the Entomological Society of America, 93, 362–366. Cognato, A. I. & Vogler, A. P. (2001). Exploring data interaction and nucleotide alignment in a multiple gene analysis of Ips (Coleoptera: Scolytinae). Systematic Biology, 50, 758 – 780. Cummings, M. P., Handley, S. A., Myers, D. S., Reed, D. L., Rokas, A. & Winka, K. (2003). Comparing bootstrap and posterior probability values in the four-taxon case. Systematic Biology, 52, 477 – 487. Damgaard, J. & Cognato, A. I. (2003). Sources of character conflict in a clade of water striders (Heteroptera: Gerridae). Cladistics, 19, 512–526. Farrell, B. D., Sequeira, A., O’Meara, B., Normark, B. B., Chung, J. & Jordal, B. (2001). The evolution of agriculture in beetles (Curculionidae: Scolytinae and Platypodinae). Evolution, 55, 2011–2027. Gibson, A., Gowri-Shankar, V., Higgs, P. G. & Rattray, M. (2005). A comprehensive analysis of mammalian mitochondrial genome base composition and improved phylogenetic methods. Molecular Biology and Evolution, 22, 251– 264. Gillespie, J. J. (2004). Characterizing regions of ambiguous alignment caused by the expansion and contraction of hairpin-stem loops in ribosomal RNA molecules. Molecular Phylogenetics and Evolution, 33, 936–943. Gillespie, J. J. (2005). Structure-based methods for the phylogenetic analysis of ribosomal RNA molecules. Unpublished PhD Dissertation. Texas A & M University, College Station. Gillespie, J. J., Cannone, J. J., Gutell, R. R. & Cognato, A. I. (2004). A secondary structural model of the 28S rRNA expansion segments D2 and D3 from rootworms and related leaf beetles (Coleoptera: Chrysomelidae; Galerucinae). Insect Molecular Biology, 13, 495–518. Gillespie, J. J., Yoder, M. J. & Wharton, R. A. (2005). Predicted secondary structure for 28S and 18S rRNA from Ichneumonoidea (Insecta: Hymenoptera: Apocrita): impact on sequence alignment and phylogeny estimation. Journal of Molecular Evolution, 61, 114 – 137. Gladstein, D. S. & Wheeler, W. C. (1997). POY: the optimization of alignment characters. Program and documentation. Available at ftp.amnh.org/pub/molecular. Gutell, R. R. (1996). Comparative sequence analysis and the structure of 16S and 23S rRNA. In A. E. Dahlberg & E. A. Zimmerman (Eds). Ribosomal RNA Structure, Evolution, Processing and Function in Protein Sysnthesis (pp. 111–129). Boca Raton, FL: CRC Press. Hudelot, C., Gowri-Shankar, V., Jow, H., Rattray, M. & Higgs, P. G. (2003). RNA-based phylogenetic methods: application to mammalian mitochondrial RNA sequences. Molecular Phylogenetics and Evolution, 28, 241–252.

Jordal, B. H. (1998). A review of Scolytodes Ferrari (Coleoptera: Scolytidae) associated with Cecropia (Cecropiaceae) in the northern Neotropics. Journal of Natural History, 32, 31– 84. Jordal, B. H. (2001). The Origin and Radiation of Sib-Mating Haplodiploid Beetles (Coleoptera, Curculionidae, Scolytinae). Bergen: University of Bergen. Jordal, B. H. (2002). Elongation Factor 1a resolves the monophyly of the haplodiploid ambrosia beetles Xyleborini (Coleoptera: Curculionidae). Insect Molecular Biology, 11, 453 – 465. Jordal, B. H. (2007). Reconstructing the phylogeny of Scolytinae and close allies: major obstacles and prospects for a solution. In B. Bentz, A. I. Cognato & K. Raffa (Eds). Proceedings from the Third Workshop on Genetics of Bark Beetles and Associated Microorganisms. Proc. RMRS-P-45 (pp. 3 – 8). Fort Collins, CO: U.S. Department of Agriculture. Forest Service, Rocky Mountain Research Station. Jordal, B. H. (2008). Scolytinae. In R. Beutel & R. A. B. Leschen (Eds). Handbook of Zoology, Band IV Arthropoda: Insecta. Part 38: Coleoptera, Beetles, Vol. 2: Morphology and systematics (Polyphaga). In press. Jordal, B. H. & Hewitt, G. M. (2004). The origin and radiation of Macaronesian beetles breeding in Euphorbia: the relative importance of multiple data partitions and population sampling. Systematic Biology, 53, 711– 734. Jordal, B. H., Beaver, R. A., Normark, B. B. & Farrell, B. D. (2002). Extraordinary sex ratios and the evolution of male neoteny in sib-mating Ozopemon beetles. Biological Journal of the Linnean Society, 75, 353 – 360. Jow, H., Gowri-Shankar, V. & Guillard, B. (2005). PHASE: a Software Package for Phylogenetics and Sequence Evolution. Program and documentation available at http://www.cs.man.ac.uk/~gowrishv/ beta-release/. Jow, H., Hudelot, C., Rattay, M. & Higgs, P. G. (2002). Bayesian phylogenetics using an RNA substitution model applied to early mammalian evolution. Molecular Biology and Evolution, 19, 1591–1601. Kjer, K. M. (1995). Use of rRNA secondary structure in phylogenetic studies to identify homologous positions: an example of alignment and data presentation from the frogs. Molecular Phylogenetics and Evolution, 4, 314 – 330. Kjer, K. M. (1997). An alignment template for amphibian 12S rRNA, domain III: conserved primary and secondary structural motifs. Journal of Herpetology, 31, 599 – 604. Kjer, K. M. (2004). Aligned 18S and insect phylogeny. Systematic Biology, 53, 506 – 514. Kjer, K. M., Gillespie, J. J. & Ober, K. A. (2007). Opinions on multiple sequence alignments, and an empirical comparison of repeatability and accuracy between POY and structural alignment. Systematic Biology, 56, 133 –146. Kruskal, J. B. (1983). An overview of sequence comparison. In D. Sankoff & J. B., Kruskal (Eds). Time Warps, String Edits, and Macromolecules: the Theory and Practice of Sequence Comparison (pp. 1– 45). Reading, MA, Addison-Wesley. Kuschel, G., Leschen, R. A. B. & Zimmerman, E. C. (2000). Platypodidae under scrutiny. Invertebrate Taxonomy, 14, 771– 805. Lee, M. S. Y. (2001). Unalignable sequences and molecular evolution. Trends in Ecology and Evolution, 16, 681– 685. Lin, C.-P. & Danforth, B. N. (2004). How do insect nuclear and mitochondrial gene substitution patterns differ? Insights from Bayesian analyses of combined datasets. Molecular Phylogenetics and Evolution, 30, 686 – 702.


55


Marvaldi, A. E., Sequeira, A. S., O’Brien, C. W. & Farrell, B. D. (2002). Molecular and morphological phylogenetics of weevils (Coleoptera, Curculionoidea): do niche shifts accompany diversification? Systematic Biology, 51, 761– 785. Mathews, D. H., Sabina, J., Zuker, M. & Turner, D. H. (1999). Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. Journal of Molecular Biology, 288, 911– 940. Morrison, D. A. (2006). Multiple sequence alignment for phylogenetic purposes. Australian Systematic Botany, 19, 479– 539. Normark, B. B., Jordal, B. H. & Farrell, B. D. (1999). Origin of a haplodiploid beetle lineage. Proceedings of the Royal Society of London Series B, 266, 2253 – 2259. Posada, D. & Crandall, K. (1998). MODELTEST: testing the model of DNA substitution. Bioinformatics, 14, 817 – 818. Rambaut, A. & Drummond, A. J. (2004). TRACER, Version 1.1. Available from http://evolve.zoo.ox.ac.uk/. Ronquist, F. & Huelsenbeck, J. P. (2003). MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics, 19, 1572–1574. Sankoff, D. (1975). Minimal mutation trees of sequences. SIAM Journal of Applied Mathematics, 28, 35 – 42. Sankoff, D. & Cedergren, R. J. (1983). Simultaneous comparison of three or more sequences related by a tree. In D. Sankoffm & J. B. Kruskal (Eds) Time Warps, String Edits, and Macromolecules: the Theory and Practice of Sequence Comparison (pp. 253 – 263). Reading, MA: Addison-Wesley. Sankoff, D., Morel, C. & Cedergren, R. J. (1973). Evolution of 5S RNA and the non-randomness of base replacement. Nature: New Biology, 245, 232 – 234. Savill, N., Hoyle, D. & Higgs, P. (2001). RNA sequence evolution with secondary structure constraints: comparison of substitution rate models using maximum likelihood methods. Genetics, 157, 399–411.

56

Sequeira, A. S., Normark, B. B. & Farrell, B. D. (2000). Evolutionary assembly of the conifer fauna: distinguishing ancient from recent associations in bark beetles. Proceedings of the Royal Society of London Series B, 267, 2359 – 2366. Sequeira, A. S., Normark, B. B. & Farrell, B. B. (2001). Evolutionary origins of Gondwanan interactions: how old are Araucaria beetle herbivores? Biological Journal of the Linnean Society, 74, 459 – 474. Swofford, D. (2002). PAUP*. Phylogenetic Analysis Using Parsimony (*and other methods) version 4. Sinauer Associates, Sunderland, Massachusetts. Wheeler, W. C. (1996). Optimization alignment: the end of multiple sequence alignment in phylogenetics? Cladistics, 12, 1– 9. Whiting, M. F., Carpenter, J. C., Wheeler, Q. D. & Wheeler, W. C. (1997). The Strepsiptera problem: Phylogeny of the insect orders inferred from 18S and 28S ribosomal DNA sequences and morphology. Systematic Biology, 46, 1– 68. Wood, S. L. (1973). On the taxonomic status of Platypodidae and Scolytidae (Coleoptera). Great Basin Naturalist, 33, 77 – 90. Wood, S. L. (1978). A reclassification of the subfamilies and tribes of Scolytidae (Coleoptera). Annales de la Sociètè Entomologique de France, 14, 95 –122. Wood, S. L. (1982). The bark and ambrosia beetles of North and Central America (Coleoptera: Scolytidae), a taxonomic monograph. Great Basin Naturalist Memoirs, 6, 1–1359. Wood, S. L. (1986). A reclassification of the genera of Scolytidae (Coleoptera). Great Basin Naturalist Memoirs, 10, 126. Wood, S. L. (1993). Revision of the genera of Platypodidae (Coleoptera). Great Basin Naturalist, 53, 259 – 281. Wood, S. L. & Bright, D. (1992). A catalog of Scolytidae and Platypodidae (Coleoptera). Part 2: taxonomic index. Great Basin Naturalist Memoirs, 13, 1–1553. Zuker, M. (2003). Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Research, 31, 3406 –3415.
