76 (1): 45 – 58 14.5.2018

© Senckenberg Gesellschaft für Naturforschung, 2018.

New molecular markers resolve the phylogenetic position of the enigmatic wood-boring Platypodinae (Coleoptera: )

Sigrid Mugu, Dario Pistone & Bjarte H. Jordal *

Department of Natural History, The University Museum, University of Bergen, PO Box 7800, NO-5020 Bergen, Norway; Sigrid Mugu [syky33­­@hotmail.com]; Dario Pistone [[email protected]]; Bjarte Jordal * [[email protected]] — * Corresponding author

Accepted 06.xii.2017. Published online at www.senckenberg.de/-systematics on 30.iv.2018. Editors in charge: Rudolf Meier & Klaus-Dieter Klass

Abstract. The precise phylogenetic position of the subfamily Platypodinae continues to be one of the more contentious issues in weevil systematics. Morphological features of adult and similar ecological adaptations point towards a close relationship with the wood boring Scolytinae, while some recent molecular studies and larval morphology have indicated a closer relationship to Dryophthori­ nae. To test these opposing hypotheses, a molecular phylogeny was reconstructed using 5,966 nucleotides from ten gene fragments. Five of these genes are used for the first time to explore phylogeny, i.e. the nuclear protein coding genes PABP1, UBA5, Arr2, TPI, and Iap2, while five markers have been used in earlier studies (28S, COI, CAD, ArgK, and EF-1α). Bayesian, maximum likelihood and parsimony analyses of the combined data strongly support a monophyletic Curculionidae (the advanced weevils with geniculate antennae), where Brachycerinae, Platypodinae, and formed the earliest diverging groups. Dryophthorinae and core Platypodinae were sister groups with high support, with the contentious genera Mecopelmus Blackman, 1944 and Coptonotus Chapuis, 1873 placed elsewhere. Other lineages of wood boring weevils such as Scolytinae, , and Conoderinae were part of a derived, but less resolved, clade forming the sister group to . Resolution among major curculionid subfamilies was ambiguous, emphasizing the need for large volumes of data to further improve resolution in this most diverse section of the weevil tree.

Key words. Weevils, molecular phylogeny, Platypodinae, Scolytinae, Dryophthorinae, ambrosia beetles, TPI, UBA5, PABP1, Arrestin2, Iap2.

1. Introduction

The weevil superfamily Curculionoidea represents one variety of older diverging lineages, including Nemony­ of the most diverse groups of , with more than chidae, Anthribidae, Attelabidae, , Caridae, and 60,000 described (Oberprieler et al. 2007). Clas­ . Most of the controversy is therefore associ­ sification of the group has changed considerably over ated with the placement and rank of the advanced wee­ the past centuries, as can be expected for such a tre­ vils which are characterized by geniculate antennae – the mendously diversified group. Recent revisions of higher megadiverse family Curculionidae sensu Oberprieler et taxa (Alonso-Zarazaga & Lyal 1999; Oberprieler et al. (2007) (Fig. 1). The generally low phylogenetic reso­ al. 2007) have highlighted considerable uncertainty tied lution obtained so far may be a consequence of limited to the placement and rank of certain taxa, but have also molecular data per taxon unit, as well as the high number pointed towards a gradually unified classification, largely of species, with species-rich clades requiring larger data founded on, and confirmed by, recent phylogenetic anal­ volumes to obtain resolution. The type of data used in yses (Kuschel 1995; Marvaldi et al. 2002; McKenna et previous analyses has mainly been of ribosomal or mito­ al. 2009; Jordal et al. 2011; Haran et al. 2013; Gillett chondrial origin, with no more than five nuclear protein et al. 2014; Gunter et al. 2015). coding genes applied to date (Farrell et al. 2001; Mc­ There is now a certain consensus that orthocerous Kenna et al. 2009; Jordal et al. 2011; Riedel et al. 2016). weevil families (weevils with straight antennae) form a A commonly used ribosomal marker, the 18S gene, has

ISSN 1863-7221 (print) | eISSN 1864-8312 (online) 45 Mugu et al.: Weevil phylogeny

Fig. 1. Two main hypotheses on relationships in the advanced weevils, using Brentidae as outgroup: A: Proposed by Kuschel (1995) and partially supported by mixed morphological and molecular data in Farrell (1998), Marvaldi et al. (2002), and Jordal et al. (2011). B: Proposed by Marvaldi (1997) and supported by molecular data in McKenna et al. (2009), Haran et al. (2013), and Gillett et al. (2014). Subfamilies marked by * as broadly defined byO berprieler et al. (2007).

a low substitution rate and, hence, contains very limited tribe Campyloscelini (Jordal et al. 2011; Kirkendall et information for weevil phylogenetics (Farrell 1998). al. 2014). Wood boring taxa have often been placed close Additional markers are therefore much needed to enable to each other in classifications due to morphological further resolution of the weevil tree. similarities (Blandford 1897; Kuschel 1995; Kuschel Perhaps the most contentious issue in weevil phylo­ et al. 2000; Oberprieler et al. 2014; see Fig. 1A), in par­ genetics is the placement of the wood boring and fungus- ticular, Platypodinae and Scolytinae (Wood 1986; Mo- farming subfamily Platypodinae (Jordal et al. 2014; Jor- rimoto & Kojima 2003). However, morphological data dal 2015). These beetles share a functional niche with (Lyal 1995; Marvaldi 1997) and recent molecular phy­ 11 fungus-farming lineages in another weevil subfamily, logenetic studies (McKenna et al. 2009; Gillett et al. Scolytinae (Hulcr et al. 2015). These all live in nutri­ 2014; Gunter et al. 2015) have indicated that this may tional symbiosis with Microascales and Ophiostomatales not reflect evolution, but adaptation to similar life styles. ambrosia fungi and are therefore generally referred to as Some large-scale molecular studies have rather suggest­ ‘ambrosia beetles’ (Beaver 1989). Platypodine and sco­ ed a close, but weakly supported, relationship between lytine ambrosia beetles excavate tunnel systems in dead Platypodinae and Dryophthorinae (McKenna et al. 2009; trees into which they inoculate fungal spores and culti­ Haran et al. 2013; Gillett et al. 2014), in particular vate small fungal gardens in the wood; this serves as the agreement with larval and pupal morphology (Marvaldi only food source for their larvae. Fungus farming is a tru­ 1997). Both types of data also suggest that platypodines ly unique evolutionary innovation seen elsewhere only in and dryophthorines are advanced weevils, forming the one clade of ants and one clade of termites (Mueller & first diverging clade after the origin of Brachycerinae Gerardo 2002). (Fig. 1B). All three subfamilies (sensu Oberprieler et al. The wood boring behaviour that characterizes bark 2007) have therefore been ranked as families by some and ambrosia beetles is generally associated with a sub­ authors, as opposed to subfamilies, and placed outside stantial reduction in rostrum length and strengthened tib­ a more narrowly defined Curculionidae (sensu Thomp- ial spines, a feature also seen in some other wood boring son 1992; Zimmerman 1993, 1994; Alonso-Zarazaga & weevils such as many Cossoninae and the conoderine Lyal 1999).

46 ARTHROPOD SYSTEMATICS & PHYLOGENY — 76 (1) 2018

In order to establish a more robust resolution in the TPI is a key enzyme of the glycolysis pathway weevil phylogeny, we added five new nuclear protein (Wierenga et al. 2010) and has occasionally been used coding genes to the phylogenetic analysis. The new in phylogenetic analyses of insects (Hardy 2007; Wieg- markers were originally screened and optimized for bark mann et al. 2009; McKenna & Farrell 2010). beetle phylogenetics (Pistone et al. 2016), and we have Arr2 is a mediator protein involved in the sensitiza­ tested their usefulness for a broader range of weevil taxa. tion of G-protein-coupled receptors and in other signal­ Based on new sequence data, we tested the hypothesis ling pathways (Gurevich & Gurevich 2006). Molecular that Platypodinae is the sister group to Dryophthorinae, characterization of this gene in Maruca vitrata (Lepido­ using a largely unbiased taxon sampling that represents ptera: Crambidae) has demonstrated congruence with ba­ most major groups of advanced weevils. sal holometabolan relationships and could potentially be valuable as a phylogenetic marker (Chang & Ramasamy 2013). Iap2 is a member of the inhibitor of apoptosis pro­ 2. Materials and methods tein family, mainly involved in regulation of caspase activity ensuring cell survival (Leulier et al. 2006; Huh et al. 2007). Iap2 in particular is required for the innate Samples included 72 species of 15 different subfamilies immune response to Gram-negative bacterial infections in the family Curculionidae, sensu Alonso-Zarazaga & (Rajalingam et al. 2006). Lyal (1999), or 9 subfamilies sensu Oberprieler et al. UBA5 is an E1 enzyme responsible for the activation (2007). Ten species of Anthribidae, Attelabidae, Api­ of ubiquitin-fold modifier 1 protein (Umf1) by forming a onidae and Brentidae were included as outgroup taxa high-energy thioester bond (Komatsu et al. 2004; Dou et (Table 2). DNA was extracted from a leg for each of the al. 2005; Bacik et al. 2010; Gavin et al. 2014). Ubiquit­ larger species, or head and pronotum for smaller species, ination, including the process of post-translational modi­ using the DNeasy® Blood & Tissue Kit (Qiagen, Hilden, fication or addition of ubiquitin to a protein, is carried Germany) following the manufacturer’s instructions. out by activation, conjugation and ligation performed by PCR (Polymerase Chain Reaction) was used to am­ three ubiquitin-modifier classes of enzymes (E1, E2 and plify gene fragments prior to Sanger sequencing. DNA E3 respectively). Information regarding UBA5 in insects sequences were obtained from ten genes, five of these is very limited. have not previously, or only rarely, been used in beetle PABP1 is known as the Poly (A) binding protein 1, phylogenetics: Triose phosphate isomerase (TPI), Arres­ which plays a crucial role for the messenger RNA trans­ tin 2 (Arr2), Inhibitor of apoptosis 2 (Iap2), Ubiquitin- portation from the nucleus (Apponi et al. 2010). This pro­ like modifier-activating enzyme 5 (UBA5), and Polyade­ tein has a conserved structure in the Metazoa (Smith et al. nylate-binding protein 1 (PABP1). 2014). PABP1 has not been used in phylogenetic studies

47 Mugu et al.: Weevil phylogeny - – – – – – – – – – – – – – – – l o ns – – – TPI A KX160555 KX160556 KU041951 KU041961 KU041962 KU041952 KU041966 KU041953 KU041967 KU041959 KU041958 KU041954 KU041956 KU041957 KU041955 KU041960 KU041963 – – – – – – – – – – – – – – – – – – – – Arrestin2 KX160643 KX160641 KU163337 KU163338 KU163348 KU163339 KU163340 KU163349 KU163350 KU163342 KU163341 KU163344 KU163353 KU163347 KU163346 – – – – – – – – – – – – – – – – – Iap2 KX160628 KU042009 KU042010 KU042021 KU042011 KU042022 KU042018 KU042029 KU042016 KU042015 KU042012 KU042023 KU042024 KU042025 KU042013 KU042014 KU042020 KU042019 – – – – – – – – – – – – – – – UBA5 KX160706 KX160704 KU041987 KU041975 KU041988 KU041982 KU041983 KU041994 KU041976 KU041995 KU041977 KU041980 KU041978 KU041990 KU041991 KU041979 KU041985 KU041984 KU041986 KU041989 – – – – – – PABP1 KX160754 KX160755 KX160751 KU041907 KU041924 KU041908 KU041909 KU041920 KU041921 KU041911 KU041930 KU041910 KU041931 KU041912 KU041932 KU041934 KU041917 KU041916 KU041926 KU041927 KU041928 KU041914 KU041915 KU041913 KU041918 KU041939 KU041922 KU041923 KU041925 – – – – – – – – – ArgK KU041879 KU041880 HQ883865 HQ883841 KU041882 KU041878 HQ883868 HQ883867 HQ883842 HQ883843 HQ883858 HQ883878 HQ883845 HQ883846 HQ883853 HQ883852 HQ883848 HQ883872 HQ883850 HQ883851 HQ883849 HQ883856 HQ883892 HQ883860 HQ883859 HQ883862 – – – – – – – – – – – – – – – – – – CAD KU041884 KU041885 HQ883778 HQ883765 KU041886 HQ883780 HQ883779 HQ883767 HQ883768 HQ883772 HQ883771 HQ883769 HQ883785 HQ883770 HQ883774 HQ883802 HQ883776 – – – – – – – 28S KU041869 KU041867 KU041870 KU041871 HQ883549 AF308350 KU041873 KU041868 AF308351 HQ883552 HQ883551 HQ883528 HQ883529 HQ883543 HQ883563 HQ883531 HQ883532 HQ883533 HQ883540 HQ883539 HQ883535 HQ883558 HQ883537 HQ883538 HQ883536 HQ883574 HQ883546 HQ883557 – – – – – – – EF-1 α EU191870 EU191871 KU041896 KU041897 KU041899 KU041898 KU041900 KU041901 HQ883696 KU041903 AF308346 HQ883717 HQ883697 HQ883710 HQ883725 HQ883698 HQ883699 HQ883700 HQ883706 HQ883702 HQ883723 HQ883704 HQ883705 HQ883703 HQ883735 HQ883711 HQ883714 HQ883722 – – – – – CO1 EU191838 EU191839 KU041889 KU041890 KU041892 KU041893 HQ883608 KU041891 AF375307 HQ883636 AY040285 HQ883609 HQ883627 HQ883610 HQ883628 HQ883649 HQ883612 HQ883613 HQ883614 HQ883621 HQ883620 HQ883616 HQ883643 HQ883618 HQ883619 HQ883617 HQ883624 HQ883663 HQ883631 HQ883642 COUNTRY Spain Cameroon Madeira Tanzania PNG Madagascar Norway Russia South Africa Russia Cameroon Norway South Africa Norway Madagascar Sarawak Madagascar Cameroon Spain Cameroon Norway Norway Cameroon Uganda Cameroon Cameroon South Africa Russia South Africa CR Panama Argentina Argentina PNG Australia CODE CsMes01 Antrib02 CsPse01 Antrib03 CsPsc01 Antrib04 CsRhy02 AtApo01 Crh_sp1 AtApo02 Crh_sp2 BrApi03 CuSib01 BrApi01 Dryoph01 BrBre01 Dryoph02 BrBre02 Dryoph05 BrBre03 CeZac01 CeRhi01 CdCod02 CsXxA01 CsXxA02 CsXxA03 CdSpr01 CdZyg01 CdMet CpCop01 MeMec01 CsAru02 CsAru01 CsCpt02 CsXen01 SPECIES fusiformis Anthribidae indet. sp2 sp. Anthribidae indet. sp3 Pseudostenocelis sp. Anthribidae indet. sp3 elongatus Rhyncolus Apoderus coryli Apoderus indet . sp1 Cryptorhynchinae Apoderus jekeli Apoderus Cryptorhynchini indet. sp2 Cryptorhynchini Apion (Apion) cruentatum (Apion) Apion sp. Apion () curtirostre (Perapion) Apion Dryophthorinae indet. sp1 Dryophthorinae Brentinae indet. sp1 Brentinae Dryophthorinae indet. sp2 Dryophthorinae Brentinae indet. sp2 Brentinae cruentatus Rhynchophorus Brentinae indet. sp3 Brentinae affinis Rhinoncus pericarpius Campyloscelini indet. Campyloscelini Homoeometamelus sp1 Homoeometamelus sp2 Homoeometamelus sp3 Scolytoproctus sp. Scolytoproctus Conoderini sp. Metialma sp. Coptonotus cyclopus Mecopelmus zeteki Araucarius major Araucarius Araucarius minor Araucarius Coptocorynus sp. Xenocnema sp. TRIBE indet. Onycholipini indet. Onycholipini indet. Apoderini Cryptorhynchinae Apoderini Cryptorhynchini Apionini Tychiini Apionini indet. (Brentinae) indet. (Brentinae) (Brentinae) Phytobiini Campyloscelini Campyloscelini Campyloscelini Campyloscelini Campyloscelini Conoderini Conoderini Coptonotini Mecopelmini Araucariini Araucariini Araucariini Araucariini et al. (1999). a r aza g FAMILY Cossoninae Anthribidae Cossoninae Anthribidae Cossoninae Anthribidae Cossoninae Attelabidae Cryptorhynchinae Attelabidae Cryptorhynchinae Apionidae Apionidae Dryophthorinae Brentidae Dryophthorinae Brentidae Dryophthorinae Brentidae Curculionidae SUBFAMILY Ceutorhynchinae Conoderinae Conoderinae Conoderinae Conoderinae Conoderinae Conoderinae Conoderinae Coptonotinae Coptonotinae Cossoninae Cossoninae Cossoninae Cossoninae Species included in this study and their GenBank accession numbers. The code represents the DNA specimen voucher at the University Museum of Bergen. The classification scheme follows scheme classification The Bergen. of Museum University the at voucher specimen DNA the represents code The numbers. accession GenBank their and study this in included 2. Species Table Z

48 ARTHROPOD SYSTEMATICS & PHYLOGENY — 76 (1) 2018 – – – – – – – – – – – – – – – – – – – – – – – – TPI KX160560 KX160569 KU041973 KU041974 KU041965 KU041964 KU041968 KX160564 KX160565 KU041969 KU041970 KU041971 KU041972 – – – – – – – – – – – – – – – – – – – – – Arrestin2 KX160647 KX160657 KX160652 KX160653 KU163357 KU163358 KU163352 KX160650 KU163351 KX160651 KU163343 KX160654 KU163345 KU163354 KU163355 KU163356 – – – – – – – – – – – – – – – – – Iap2 KX160625 KX160637 KU042039 KX160638 KU042040 KU042028 KU042027 KX160634 KU042026 KU042031 KU042032 KU042030 KX160635 KU042017 KU042033 KU042034 KU042035 KU042036 KU042037 KU042038 – – – – – – – – – – – – – UBA5 KX160709 KX160721 KX160705 KX160717 KX160718 KU042007 KU041997 KX160719 KU042008 KU041996 KU041993 KU041992 KU041998 KX160714 KX160715 KU041981 KU041999 KU042000 KU042001 KU042002 KU042003 KU042004 KU042005 KU042006 – – – – – – – PABP1 KX160760 KX160773 KX160774 KX160752 KX160769 KX160770 KU041949 KX160771 KU041950 KU041933 KX160765 KU041936 KU041929 KU041937 KU041935 KX160766 KU041938 KX160767 KU041940 KX160775 KU041941 KU041942 KU041919 KU041943 KU041944 MF771641 KU041945 KU041946 KU041947 KU041948 – – – – – – – – – ArgK JX263918 KU041881 KU041883 HQ883920 HQ883883 HQ883874 HQ883886 HQ883884 HQ883887 HQ883882 HQ883888 HQ883911 HQ883854 HQ883907 HQ883855 HQ883908 HQ883893 HQ883909 HQ883895 HQ883894 HQ883896 HQ883857 HQ883904 HQ883912 HQ883914 HQ883915 HQ883916 HQ883918 – – – – – – – – – – – – CAD KU041887 KU041888 HQ883834 HQ883788 HQ883795 HQ883793 HQ883796 HQ883797 HQ883824 HQ883825 HQ883773 HQ883818 HQ883819 HQ883803 HQ883820 HQ883805 HQ883804 HQ883806 HQ883775 HQ883814 HQ883826 HQ883828 HQ883829 HQ883830 HQ883832 – – – – – 28S AF308365 KU041877 KU041874 KU041872 KU041875 KU041876 HQ883602 HQ883567 HQ883560 HQ883570 HQ883568 HQ883566 HQ883571 HQ883591 HQ883569 HQ883592 HQ883541 HQ883587 HQ883562 HQ883588 HQ883575 HQ883589 HQ883577 HQ883576 HQ883578 HQ883542 HQ883583 HQ883593 HQ883595 HQ883596 HQ883597 HQ883599 – – – – – – – – EF-1 α AF439742 AF308409 KU041906 KU041902 KU041904 EU191897 KU041905 HQ883760 HQ883728 HQ883731 HQ883729 HQ883727 HQ883749 HQ883730 HQ883750 HQ883707 HQ883745 HQ883708 HQ883736 HQ883746 HQ883737 HQ883738 HQ883709 HQ883743 HQ883751 HQ883753 HQ883754 HQ883755 HQ883757 – – – – – – – CO1 AF438508 KU041894 EU191865 HQ883691 HQ883652 HQ883645 HQ883656 HQ883653 HQ883657 HQ883651 HQ883658 HQ883680 HQ883654 HQ883681 HQ883622 HQ883676 HQ883623 KU041895 HQ883664 HQ883677 HQ883666 HQ883665 HQ883667 HQ883626 HQ883674 HQ883682 HQ883684 HQ883685 HQ883686 HQ883688 COUNTRY PNG Norway CR Madagascar Norway Norway Cameroon Norway Ghana Norway Sweden Russia Guyana Norway USA Russia Norway Russia Guyana Russia Guyana South Africa Sweden South Africa Norway Cameroon PNG Tanzania CR CR Cameroon CR Cameroon PNG PNG Sarawak Australia NSW Australia CODE TsSpa01 Dryoph06 TsTes01 Dryoph04 DrDry02 EnOti01 CtMic03 EnOti02 HlDac01 EnPol01 HlHyl02 EnChl01 HlPhb02 EnSit01 ToDen02 ErHim01 ToTom01 ClLar01 ScCam02 ClLix01 ScCne01 MoAmo01 ScScl02 MoPor01 MoHyl01 MoXxx01 Crh_Psx01 PlPla07 PlTel01 PlTel02 PlTri02 TsCen01 TsCha02 TsDia01 TsDia02 TsGen02 TsNot01 SPECIES Spathidicerus nobilis oryzae Tesserocerus ericerus Tesserocerus Sitophilus sp. Dryocoetes alni auropunctatus Micoborus angustus Otiorhynchus sulcatus Otiorhynchus Dactylipalpus grouvellei cervinus Polydrusus Hylesinus varius Chlorophanus sibiricus Chlorophanus Phloeoborus sp. lineatus Dendroctonus terebrans Dendroctonus Himasthlophallus flagellifer Tomicus piniperda Tomicus sp. Camptocerus aenipennis sp. Cnemonyx vismiaecolens Cnemonyx Amorphocerus rufipes Scolytus intricatus Porthetes hispidus Porthetes piceus indet . sp. Psepholax sp. Platypus impressus sp1 Teloplatypus sp2 Teloplatypus Triozastus marshalli Triozastus Cenocephalus sp. Chaetastus tuberculatus Diapus pusillimus Diapus unispineus Genyocerus exilis Genyocerus Notoplatypus elongatus TRIBE Tesserocerini Sitophilini Tesserocerini Sitophilini Dryocoetini Otiorhynchini Hexacolini Otiorhynchini Hylesinini Polydrusini Hylesinini Sitonini Hylesinini Sitonini Hylurgini Erirhinini Hylurgini Lixini Scolytini Lixini Scolytini Amorphocerini Scolytini Amorphocerini indet. Psepholacini Platypodini Platypodini Platypodini Platypodini Tesserocerini Tesserocerini Tesserocerini Tesserocerini Tesserocerini Tesserocerini SUBFAMILY Platypodinae Dryophthorinae Platypodinae Dryophthorinae Scolytinae Entiminae Scolytinae Entiminae Scolytinae Entiminae Scolytinae Entiminae Scolytinae Entiminae Scolytinae Erirhininae Scolytinae Scolytinae Lixinae Scolytinae Molytinae Scolytinae Molytinae Molytinae Molytinae Molytinae Platypodinae Platypodinae Platypodinae Platypodinae Platypodinae Platypodinae Platypodinae Platypodinae Platypodinae Platypodinae Table 2 continued. Table

49 Mugu et al.: Weevil phylogeny

of invertebrates, but has been briefly considered for such Table 3. The number of species successfully sequenced per fam­ analyses in vertebrates (Fong & Fujita 2011). ily or subfamily. Sequencing success higher than 50% is marked Primers and protocols for the five new gene frag­ in grey. ments were recently developed for Scolytinae (Table 1; Family – subfamily # species UBA5 PAPB1 Iap2 Arr2 TPI see also Pistone et al. 2016). Five additional markers Anthribidae 3 1 2 3 2 1 previously used in weevils were also included (Jordal et Attelabidae 2 0 1 0 1 0 al. 2011): arginine kinase (ArgK), carbamoyl-phosphate Brentidae / Apionidae 5 3 4 0 2 1 synthetase 2 - aspartate transcarbamylase - and dihy­ Brachycerinae – Erirhininae 1 0 1 0 0 0 droorotase (CAD), elongation factor 1 alpha (EF-1α), the – Ceutorhynchinae 2 1 2 2 0 2 large nuclear ribosomal subunit (28S rDNA), and the mi­ Baridinae – Conoderinae 7 4 6 6 4 4 tochondrial gene cytochrome oxidase I (COI). Coptonotinae 2 0 2 0 2 1 Nucleotide sequences were blasted in GenBank for Cossoninae 8 7 6 5 4 3 verification (minimum E value threshold = 1E-4). Or­ Curculioninae 1 0 1 0 0 0 thology was assessed in OrthoDB (Zdobnov et al. 2017) Dryophthorinae 5 4 4 2 0 3 and each of the five novel markers was analysed phylo­ Entiminae 5 1 3 2 0 0 genetically and compared to the combined result of five Molytinae 4 4 4 4 1 3 established markers (see also Pistone et al. 2016). Se­ Molytinae – Cryptorhynchinae 3 2 3 1 1 2 quences were aligned using ClustalX in BioEdit (Hall Molytinae – Lixinae 2 2 1 2 1 0 1999) and MAFFT (Katoh & Standley 2013) applying Platypodinae 12 9 11 6 7 6 default settings. Gblocks (Castresana 2000) was used to Scolytinae 10 7 8 5 6 5 reduce the number of ambiguous sites in the 28S rDNA Total sequencing success 72 45 59 38 31 31 alignment. Settings in Gblocks allowed less strict flank­ ing positions, gap positions within blocks, and small fi­ nal blocks. In the final matrix, the introns were removed likelihood and posterior values) were visualized in the from all the protein coding genes before the phylogenetic software Tracer1.6 (Rambaut et al. 2014). analysis. Maximum likelihood analyses were performed by the Phylogenetic analyses were made in a Bayesian sta­ software IQTREE (Trifinopoulos et al. 2016). Substitu­ tistical framework, or by maximum likelihood, or the tion models for each partition were selected using Model principle of parsimony. Four analyses were based on Finder (Kalyaanamoorthy et al. 2017) integrated in the concatenated datasets created for 72 taxa: i) a nucleo­ software. Node support was assessed by 1,000 bootstrap tide matrix combining 10 gene fragments (5,966 char­ replicates. acters) and divided into seven partitions (28S, COI by Parsimony analyses were made in PAUP* (Swofford coding position, and all nuclear coding genes combined 2002) with 1,000 random addition replicates and TBR by codon position); ii) the same dataset partitioned by 10 branch swapping. Gaps were treated as missing data and genes; iii) including the same 10 genes, but with third all characters were either equally weighted or third posi­ codon positions excluded and the remaining data divided tions were excluded. To assess node support, a total of into 5 partitions (28S, COI first and second positions, and 200 bootstrap replicates were performed, using 10 ran­ other protein coding genes first and second positions); dom addition replicates per bootstrap replicate. iv) a concatenated amino acid matrix (1,792 characters), Phylogenetic trees were visualized in FigTree1.3 with nine partitions divided by gene (ribosomal DNA (Rambaut & Drummond 2009) and edited in TreeGraph2 excluded). Additional Bayesian analyses were made of (Stover & Muller 2010). single genes, or combinations of these. For the Bayesian analyses, MrModeltest v. 2.3 (Po- sada & Crandall 1998) was used to determine the best substitution model for each partition based on the Akaike 3. Results information criterion (AIC). The best model for each of the ten genes, and for each codon position, was the general time reversible model with gamma distributed A total of 204 sequences were obtained for the five new rates and a proportion of invariable sites (GTR+I+Γ). markers, and 45 additional sequences were obtained for The mixed model was used for amino acid data. The five previously established markers (Tables 2, 3). PABP1 analyses were implemented in MrBayes 3.2.6 (Ron- generated readable sequences in 81.9% of the samples, quist & Huelsenbeck 2003), via the CIPRES Science followed by UBA5 (62%), Iap2 (53%), TPI (43%), and Gateway portal (Miller et al. 2011). Two sets of four Arr2 (43%). Markov Chain Monte Carlo (MCMC) chains (one cold The majority of failures were caused by negative and three heated with default temperature parameter 0.2) PCR amplification (126 of 146 failures). The most fre­ were run for 20,000,000 generations (for amino acid data quent problem with erroneous sequences was either un­ 30,000,000) and sampled every 1,000 generations. The readable or chimeric chromatograms (17/146), especially first 25% trees were discarded as burn-in, to obtain a fi­ for UBA5, PABP1, and Iap2. Only three sequences of nal tree sample of 15,000 trees. Analysis parameters (e.g. TPI were from other organisms – one nematode and two

50 ARTHROPOD SYSTEMATICS & PHYLOGENY — 76 (1) 2018

Fig. 2. Examples of length variable regions in the Iap2 and Arr2 genes, framed by red boxes. A: Iap2 alignments of nucleotides and amino acids (the first of two variable regions).B : Arr2 alignments of nucleotides and amino acids. fungi. Sequences of PABP1 and UBA5 were relatively 3.1.2. Arrestin2 – Arr2. Sequences were obtained from easy to align, whereas the amplified fragments of Arr2, 31 of the 72 species (43%). The amplified gene fragment Iap2 and TPI were more problematic due to long introns consisted of three exons and two introns. The beginning (Electronic Supplement Table S1), but also because of of the second exon contained indels that translated into one or two length-variable coding regions (Fig. 2). Intron a variable number of amino acids (Fig. 2). One triplet boundaries followed the general GT-AG rule in all genes, insertion occurred in the fourth exon only in Micro­ except for TPI in two species of Diapus Chapuis, 1865 borus angustus Jordal, 2017. The first intron varied from which had the first intron boundaries defined by GC-AG. 50 – 154 bp and was present in the majority of the ad­ vanced weevil species with the exclusion of Coptonotus and one Conoderinae species, and was absent in Brenti­ 3.1. Characteristics of new phylogenetic dae, Attelabidae, and Anthribidae. The second intron was markers 49 – 201 bp long, and occurred in the majority of taxa as described for the first intron. The third intron was 47 – 84 3.1.1. Inhibitor of apoptosis 2 – Iap2. A total of 38 good bp long and present in the majority of the sequences, with quality sequences (clearly defined peaks in the chroma­ the exclusion of 4 species (one Anthribidae, Mecopelmus togram) (52.7%) were obtained for this marker. The am­ zeteki Blackman, 1944, M. boops, and Hylesinus varius plified fragment contained in most cases two exons and (Fabricius, 1775). one intron (Electronic Supplement Table S1). The intron length varied from 50 – 274 bp, and was present in most 3.1.3. Polyadenylate-binding protein 1 – PABP1. Se­ species except Anthribidae, one Molytinae, one Dryo­ quences were obtained from 59 species (81.9%). The phthorinae, and two Platypodinae. The first exon of the total length was 441 bp, which translates into 147 ami­ amplified gene fragment contained two length-variable no acids. One triplet deletion occurred in Tesserocerus regions, resulting in 208 – 222 amino acids. These length ericius Blandford, 1895, resulting in one amino acid variable regions contained long serine repeats that were shorter fragment. Only one of the 58 successful samples difficult to align; hence they were tentatively included or contained an intron (Anthribidae, 156 bp). excluded in the phylogenetic analyses.

51 Mugu et al.: Weevil phylogeny

Table 4. Posterior probability (pp), parsimony bootstrap support (P-bs) and maximum likelihood bootstrap support (MLbs) from analyses of data set II – IV, for selected groups of weevils supported by data set I (Fig. 3). Bootstrap support values below 50 and posterior prob­ abilities below 0.95 are not shown.

II: by gene III: 3rd excluded IV: amino acids pp P-bs MLbs pp P-bs MLbs pp P-bs MLbs A: Platypodinae + Dryophthorinae 1.0 – 69 1.0 < 50 82 < .95 < 50 89 B: Dryophthorinae 1.0 71 100 1.0 77 90 1.0 < 50 100 C: Platypodinae, excl. Mecopelmus 1.0 96 100 1.0 100 100 < .95 86 97 D: Curculionidae, excl. Mecopelmus 1.0 – 81 1.0 74 98 < .95 < 50 77 E: Curculionidae w/ pedal aedeagus – < 50 50 – – – < .95 < 50 – F: Entiminae < .95 56 98 1.0 55 60 < .95 < 50 74

3.1.4. Ubiquitin-like modifier-activating enzyme 5 – Molytinae, Ceutorhynchinae, Cryptorhynchinae, Curcu­ UBA5. A total of 45 sequences (62%) were obtained for lioninae, Cossoninae, Conoderinae, and Lixinae (Baridi­ UBA5, consisting of two exons and one intron. The total nae). Each of the last five subfamilies was paraphyletic as length of the alignment was 348 bp without the intron. defined byO berprieler et al. (2007), while many smaller The length of the intron ranged from 50 – 177 bp, but clades were consistent with the Alonso-Zarazaga & was absent in Zacladus Reitter, 1913, Microborus, and Lyal (1999) subfamily system. A near-identical topology one Brentidae. One of the Platypodinae species (Diapus was found by maximum likelihood using the same 7 par­ pusillimus Chapuis, 1865) had one amino acid insertion titions in IQTREE (Electronic Supplement Fig. S1). in the second exon, resulting in one amino acid longer With the same data partitioned by gene (analysis II), peptide (116 aa). Curculionidae was monophyletic, albeit with Mecopel­ mus and Apion Herbst, 1797 forming a basal polytomy 3.1.5. Triose phosphate isomerase – TPI. Among the (Electronic Supplement Fig. S2). The tree topology was 39 samples that successfully amplified (54%), only 31 largely congruent with that based on the 7-partitions provided validated sequences of sufficient quality (43%). analysis, but notably with Himasthlophallus as sister to The amplified gene fragment contained up to three in­ Entiminae. trons separating four exons, with 564 bp translated into In the parsimony analysis of all nucleotides un­ 188 amino acids. The sequence of Scolytoproctus Faust, weighted, Curculionidae was recovered as paraphyletic 1895 (Conoderinae sensu Alonso-Zarazaga & Lyal with respect to Apion and Apoderus Olivier, 1897, with 1999) had one triplet deletion in the fourth exon. The first bootstrap support of 76 (Electronic Supplement Fig. S2). intron was 50 – 795 bp long, and was present in most spe­ Excluding the third codon position from the protein cod­ cies. The second intron was present only in one species, ing genes (analysis III) did not result in greater resolu­ Apion curtirostre Germar, 1817 and was 116 bp long. tion, or higher node support for relationships between This additional intron was not observed in our previous subfamilies, in either the Bayesian or the parsimony screening on bark and ambrosia beetles and other weevils analyses (Electronic Supplement Fig. S3). A close affin­ (Pistone et al. 2016). The third intron was 54 – 246 bp ity between the core Platypodinae and Dryophthorinae long, and was present in all amplified taxa, except Z. af­ was again confirmed with maximum support (PP = 1), finis (Ceutorhynchinae). whereas Mecopelmus grouped with Apion. In the par­ simony analysis with third positions excluded (III), En­ timinae was recovered as monophyletic (BS = 55), while 3.2. Phylogenetic analyses in the Bayesian tree, Sitona Germar, 1817 (Entiminae) was nested inside Dryophthorinae. The Bayesian and parsimony analyses of the combined The analysis of the amino acid translated data (anal­ nucleotide data produced largely congruent results for ysis IV) resulted in very similar tree topologies in the major weevil clades (Table 4). Exclusion or inclusion of parsimony and the Bayesian analyses (Electronic Sup­ the indel-rich coding regions in Arr2 and Iap2 did not plement Fig. S4). Both analyses recovered each of the change the reconstruction of these clades. subfamilies Platypodinae (ex Mecopelmus) and Dryoph­ The Bayesian analysis partitioned by codon position thorinae as monophyletic and as sister clades (PP = 0.56); per genome and 28S (7 partitions, analysis I) resulted in these two groups formed the sister lineage to all other a paraphyletic Curculionidae with respect to Mecopel­ Curculionidae (PP = 0.51). Among the latter, a monophy­ mus zeteki (Fig. 3). A sister relationship between the core letic Entiminae (PP = 0.62) formed the sister group to the Platypodinae and Dryophthorinae was maximally sup­ remaining taxa, but with a marginal posterior probability ported (PP = 1) and these two lineages formed a strongly of 0.56. In the parsimony analysis of these data, Sitona supported sister group to all other Curculionidae. The Er­ (Entiminae) grouped together with parts of Baridinae, irhininae (Brachycerinae) genus Himasthlophallus Zhe­ and Mecopelmus grouped with Entiminae. A moderately ri­khin & Egorov, 1990 formed a weakly supported sister supported clade (PP = 0.92) included taxa of Molytinae, group to Entiminae and a clade consisting of Scolytinae, Cossoninae, Scolytinae, Curculioninae, Baridinae, and

52 ARTHROPOD SYSTEMATICS & PHYLOGENY — 76 (1) 2018

Fig. 3. Phylogenetic consensus tree of dataset I, divided into seven partitions (by codon position in mitochondrial and nuclear genes, and 28S). Bayesian posterior probability values are shown above nodes, and parsimony bootstrap values below nodes.

53 Mugu et al.: Weevil phylogeny

Coptonotus, forming largely a polytomy. Conoderinae prone to wood boring adaptations, supports a sister re­ was monophyletic (PP = 0.79) and was closely related lationship between Platypodinae and Dryophthorinae to the molytine tribe Amorphocerini (PP = 0.54). In at the base of Curculionidae (Marvaldi 1997). A more both the Bayesian and parsimony analyses, Coptonotus detailed review of the historical development of morpho­ grouped together with part of a paraphyletic Scolytinae. logy-based classifications of Platypodinae and Scolyti­ Separate analyses of the five established markers nae can be found in Jordal (2014). combined, and the five new markers combined, resulted Our molecular data corroborate recent studies that in less resolved tree topologies compared to the analyses excluded Mecopelmus from Platypodinae, supporting a of all data (Electronic Supplement Fig. S5). In each case more narrowly defined subfamily that corresponds to the Curculionidae was monophyletic. The most significant core Platypodidae sensu Wood (1993) or Platypodinae difference between the two smaller datasets was a sister sensu Jordal (2015). This is generally consistent with relationship between Platypodinae and Dryophthorinae morphological characters, in particular the male genita­ that was supported by the new markers only (PP = 0.95). lia and associated abdominal structures, which are very In the nucleotide analyses of individual genes (Electronic different in Mecopelmus (see Thompson 1992; Kuschel Supplement Figs. S6 – S9), the Platypodinae-Dryophthor­ et al. 2000; Jordal 2014). Larvae are unfortunately not inae clade was supported by the ArgK and UBA5 data, known for this genus, which could potentially have clari­ and nearly so by the TPI data. All Dryophthorinae were fied the relationship to other weevil groups. The position lacking Arr2 data, while Iap2 indicated a more derived of Mecopelmus therefore appears to be one of the major position for Dryophthorinae, separate from Platypodinae. remaining challenges in weevil phylogenetics, and re­ Amino acid translated data revealed largely paraphyletic quires considerably more sequence data to solve. groups for most of the genes, except COI, Arr2, TPI, and Several molecular studies have indicated that Platypo­ Iap2, which were all monophyletic for Platypodinae, dinae and Dryophthorinae are, together with members of whereas TPI grouped Platypodinae and Dryophthorinae the Brachycerinae, distinct basal lineages in Curculioni­ as sister groups (Electronic Supplement Fig. S7). dae (McKenna et al. 2009; Gillett et al. 2014). The split between these three groups and the remaining Curculio­ nidae (including Entiminae) is supported by major dif­ ferences in the male genitalia – with Entiminae and other 4. Discussion derived Curculionidae having a pedal form as opposed to the ancestral pedotectal type seen in Dryophthorinae and Brachycerinae (Thompson 1992). The male genitalia 4.1. Weevil relationships of Platypodinae are highly reduced and therefore diffi­ cult to assess, but they have tentatively been associated This study provides the clearest evidence to date for a with the more primitive type of genitalia. Molecular data sister relationship between Dryophthorinae and the core strongly support the assertion that the platypodine aedea­ Platypodinae (sensu Jordal 2015). Previous molecular gus is derived from the pedotectal type. Brachycerinae, studies have suggested similar topologies, but these had Dryophthorinae and Platypodinae are ranked as subfami­ generally lower node support, including for this particu­ lies in the Oberprieler et al. system (2007), while given lar node (McKenna et al. 2009; Gillett et al. 2014). With full family status in the Alonso-Zarazaga & Lyal sys­ maximum support in the various analyses presented here, tem (1999). In light of the recent phylogenetic results, it seems prudent to conclude that these two subfamilies it is understandable that such discrepancies in rank oc­ are indeed sister groups. Our molecular data therefore cur. Without defined auxiliary criteria, such as the time refute a close relationship between Scolytinae and Platy­ banding criterion (Vences et al. 2013), the rank seems podinae which has been proposed repeatedly over the last largely subjective. A reconciled solution would need ad­ centuries (Blandford 1897; Schedl 1972; Wood 1978; ditional information on the Brachycerinae in particular, a Kuschel 1995; Kuschel et al. 2000; Bright 2014), even group which may consist of multiple unrelated lineages in mixed molecular- and morphology-based analyses (McKenna et al. 2009; Gillett et al. 2014) and, hence, (Marvaldi et al. 2002; Jordal et al. 2011). will be simultaneously affected by changes in the rank of Previous comparative analyses of morphological data Dryophthorinae and Platypodinae (see also Jordal et al. focussed to a large extent on adult head structures (Wood 2014). 1978, 1986; Morimoto & Kojima 2003), features that Our study also confirms a long-standing hypothesis are heavily modified through adaptation to wood boring that Entiminae form part of a distinct lineage of broad- and therefore not necessarily homologous in taxa with nosed weevils placed among the more advanced Cur­ similar feeding behaviour (e.g. Lyal 1995). Several other culionidae. Data on mitochondrial genomes have also features associated with wood tunnelling show extensive shown that Cyclominae and (sensu Alonso- homoplasy, including the shape of legs with hooks and Zarazaga & Lyal 2009) belong to this lineage (Gillett denticles used for substrate attachment, for instance in et al. 2014; Gunter et al. 2015). Together they form the unrelated groups such as Campyloscelini (Conoderinae) sister group to all other advanced weevils, including Cos­ and in Araucariini (Cossoninae, see e.g. Kuschel 1966; soninae, Scolytinae, a broadly defined Molytinae, Curcu­ Jordal et al. 2011). Larval anatomy, which may be less lioninae, and Baridinae (see also McKenna et al. 2009).

54 ARTHROPOD SYSTEMATICS & PHYLOGENY — 76 (1) 2018

The advanced weevil clade also includes the genus Cop­ Low resolution could also be due to missing data, tonotus, which therefore has a very distant relationship particularly in TPI, Arr2, and Iap2, which were prob­ to Mecopelmus – both of which have been placed in the lematic to amplify across all Curculionoidea. These gene same family Coptonotinae by some authors (e.g. Schedl fragments sometimes contained very long introns that 1962; Wood 1993; Wood & Bright 1992). Molecular may require further optimization of PCR extension times data were indecisive in placing Coptonotus which seems and improved primer design. Furthermore, some primers to be an old isolated lineage consisting of only four appear to be taxon specific, such as Iap2, which mainly known species (Smith & Cognato 2016). amplified species of Anthribidae, Molytinae, Baridinae, The limited resolution of the major lineages of ad­ and Cossoninae; TPI, which mainly amplified species vanced weevils is not very surprising given the enormous of Molytinae, Baridinae and Dryophthorinae; while the diversity characterising this part of the weevil tree. Rela­ Arr2 and TPI primers did not amplify any Entiminae. tionships among Curculioninae, Molytinae, and Baridinae The same three genes were also problematic to align, in (sensu Oberprieler et al. 2007) were largely unresolved part due to the irregular length of introns, and in Iap2 and also in previous molecular studies, including those based Arr2 this was also due to length variable coding regions. on mitochondrial genomes (Haran et al. 2013; Gillett et These length variable regions may be informative for cer­ al. 2014). Most of the incongruence found in our study is tain clades (Pistone et al. 2018), but their signature varies mainly associated with the deepest nodes in each of these considerably among weevil taxa and is generally known subfamilies, reflecting potential problems with the broad to be rather homoplasious across families and orders of concept of classification proposed by Oberprieler et al. insects (Ajawatanawong & Baldauf 2013; Hardy 2007). (2007). The Alonso-Zarazaga & Lyal (1999) system is Incongruence of single genes may also contribute to on the other hand more finely divided into many more reduced resolution in the weevil tree topology. The single subfamilies and each of these is therefore less likely to most deviant gene in this respect was Iap2, which placed be polyphyletic. Consistent with the latter system we re­ Dryophthorinae in a highly supported derived position covered separate clades for the ‘baridine’ groups Ceuto­ separate from Platypodinae. However, this strong sup­ rhynchinae and Conoderinae, and separate clades for the port faded when the data were translated to amino acids ‘molytine’ groups Lixinae, Cryptorhynchinae, and Mo­ and became more similar in topology to the TPI data. lytinae sensu stricto. However, our taxonomic sampling There is a slight possibility that some of the genes in­ was limited to just a few genera for each of these groups clude a mixture of multiple gene copies, which is known and can therefore not provide a proper test of monophyly. for some genes such as Elongation Factor 1-alpha in bark A recent molecular study on Cryptorhynchinae illustrat­ beetles (Jordal 2002). Different copies of this gene can ed, for instance, the many problems with placing atypical nonetheless be detected by different intron structure and members of ‘molytine’ subgroups (Riedel et al. 2016). highly divergent sequences, but were not observed in our dataset. Among the other 9 genes we could not detect any signs of paralogous copies based on OrthoDB analyses 4.2. Application of novel molecular using all available Coleoptera and Hymenoptera sequenc­ markers es. It is therefore not very likely that paralogous copies are responsible for the observed incongruence across in­ The optimization and application of five new molecular dividual genes. Rather, it is anticipated that single genes markers in weevil phylogenetics was promising despite are not able to provide phylogenetic signals that corre­ a variable degree of PCR amplification. A modest in­ spond to comprehensive multi-gene analyses (McKenna crease in new molecular data – less than doubling the et al. 2009; Gillet et al. 2014). Instead, we observed a number of nucleotides – gave increased node support significant increase in resolution and node support with for the Dryophthorinae-Platypodinae clade in particular, a stepwise addition of five new markers. The clearest in­ but also in the node connecting Scolytinae, Cossoninae, dication of such accumulative effects from the new data Curculioninae, and the broadly defined Baridinae and was the better resolution of the core Platypodinae, which Molytinae (compared to McKenna et al. 2009; Gillett was monophyletic or nearly so for Arr2, UBA5, Iap2, and et al. 2014). Several deeper nodes on the other hand ap­ TPI, while only 28S among the established markers sup­ peared to conflict with well-established topologies, indi­ ported monophyly of the subfamily. cating high substitution rates in many of these markers. To enable a more complete resolution in the phylo­ They therefore seem to have limited potential in resolv­ geny of main weevil groups, larger volumes of genomic ing older weevil relationships (see Pistone et al. 2016). data are required. New data are currently being processed More­over, we obtained low resolution in the most di­ as a part of the 1-Kite project where a broadly sampled verse clade of Curculionidae, similar to recent phyloge­ weevil phylogeny will be reconstructed from more than netic studies based on complete or partial mitochondrial 1,000 loci (McKenna et al. unpubl. data) obtained by genomes (Haran et al. 2013; Gillett et al. 2014; Gunter anchored hybrid enriched sequence capture (Lemmon et et al. 2015). In general, it appears difficult to obtain reso­ al. 2012). This approach will likely become the stand­ lution in this most diverse section of the weevil tree, and ard procedure in large scale phylogenetics in the future, is likely a consequence of high diversity, involving tens which could make PCR-based Sanger sequencing redun­ of thousands of species (Oberprieler et al. 2007). dant (Brady et al. 2014; Faircloth et al. 2015). Howev­

55 Mugu et al.: Weevil phylogeny

er, most phylogenies made in connection with taxonomic Faircloth B.C., Branstetter M.G., White N.D., Brady S.G. 2015. work are more practically obtained with smaller data Target enrichment of ultraconserved elements from volumes. Given that PCR amplification of few genes and provides a genomic perspective on relationships among Hyme­ noptera. – Molecular Ecology Resources 15: 489 – 501. individuals is still much faster and cheaper than next gen­ Farrell B.D. 1998. “Inordinate fondness” explained: Why are there eration sequencing, the Sanger method will still be need­ so many beetles? – Science 281: 555 – 559. ed for small-scale routine phylogenetics such as DNA Farrell B.D., Sequeira A.S., O’Meara B.C., Normark B.B., barcoding and integrative . Thus, our twofold Chung J.H., Jordal B.H. 2001. The evolution of agriculture in aim here was to develop primers and protocols for new beetles (Curculionidae: Scolytinae and Platypodinae). – Evolu­ tion 55: 2011 – 2027. molecular markers, and to use the new data to test one Fong J.J., Fujita M.K. 2011. Evaluating phylogenetic informa­ particularly interesting relationship – the one between tiveness and data-type usage for new protein-coding genes Dryophthorinae and Platypodinae. We believe the new across Vertebrata. – Molecular Phylogenetics and Evolution 61: data obtained have demonstrated considerable promise in 300 – 307. achieving these aims. Gavin J.M., Hoar K., Xu Q., Ma J., Lin Y., Chen J. et al. 2014. Mechanistic study of Uba5 enzyme and the Ufm1 conjugation pathway. – Journal of Biological Chemistry 289: 22648 – 22658. Gillett C.P.D.T., Crampton-Platt A., Timmermans M.J.T.N., Jor- dal B.H., Emerson B.C., Vogler A.P. 2014. Bulk de novo mi­ 5. 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Electronic Supplement Files at http://www.senckenberg.de/arthropod-systematics

File 1: mugu&al-curculionidaephylogeny-asp2018-electronicsup plement-1.pdf — Fig. S1. Phylogeny resulting from the maximum likelihood analysis I in IQTree, divided into seven partitions (by codon position in mitochondrial and nuclear genes, and 28S). Boot­ strap support values are shown on nodes. — Fig. S2. Phylogeny resulting from the Bayesian analysis of dataset II, divided into ten partitions (by gene). Posterior probability values above the nodes, and parsimony bootstrap values below. — Fig. S3. Phylogeny re­ sulting from the Bayesian analysis of dataset III, divided into five partitions (by 28S, and genome and codon positions with third positions excluded). Posterior probability values above the nodes, parsimony bootstrap values below. — Fig. S4. Phylogeny result­ ing from the Bayesian analysis of amino acid data (dataset IV), divided into nine partition by gene. Posterior probability values above the nodes, parsimony bootstrap values below. — Fig. S5. Combined analysis of five gene fragments. A: Phylogeny based on Bayesian analysis of EF-1α, CO1, 28S, CAD and ArgK. B: Based on TPI, UBA5, Arr2, Iap2 and PABP1. — Fig. S6. Tree topologies resulting from the individual Bayesian analyses of PABP1, UBA5, Arr2, Iap2 and TPI. — Fig. S7. Tree topologies resulting from the individual Bayesian analyses of amino acid translated data from PABP1, UBA5, Arr2, Iap2 and TPI. — Fig. S8. Tree topologies resulting from the individual Bayesian analyses of COI, EF-1α, CAD, 28S and ArgK. — Fig. S9. Tree topologies resulting from the individual Bayesian analyses of amino acid translated COI, EF- 1α, CAD and ArgK. File 2: mugu&al-curculionidaephylogeny-asp2018-electronicsup plement-2.doc — Table S1. Description of the length of the coding sequence in terms of translated amino acids (aa) and the number of intervening introns. Voucher codes refer to taxa listed in Table 2.

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