Front. Ecol. Evol. Frontiers in Ecology and Evolution Front. Ecol. Evol. 2296-701X Frontiers Media S.A. 10.3389/fevo.2013.00001 Ecology and Evolution Original Research Article Molecular phylogeny and taxonomic review of Premnobiini Browne, 1962 (Coleoptera: Curculionidae: Scolytinae) Cognato Anthony I. * Department of Entomology, Michigan State University East Lansing, MI, USA

Edited by: Alejandro P. Rooney, U.S. Department of Agriculture, USA

Reviewed by: Bjarte H. Jordal, University of Bergen, Norway; Jiri Hulcr, University of Florida, USA

*Correspondence: Anthony I. Cognato, Department of Entomology, Michigan State University, 288 Farm Lane Road, East Lansing, MI 48824, USA e-mail: cognato@msu.edu

This article was submitted to Phylogenetics, Phylogenomics, and Systematics, a section of the journal Frontiers in Ecology and Evolution.

 04 11 2013 29 11 2013 2013 1 1 29 10 2013 17 11 2013 Copyright © 2013 Cognato. 2013

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

The taxonomy of Premnobiini is reviewed in the context of a molecular phylogeny including species of Ipini, Dryocoeotini, and Xyleborini. DNA data from COI, 16S, 28S, and CAD (~2640 characters) were generated for 79 species. Parsimony and Bayesian methods, using multiple sequence alignment methods and partitioning regimes, were used to reconstruct the phylogeny. The resulting topologies are generally congruent. Ipini is monophyletic along with all genera except Acathotomicus. Premnobiini is nested within Ipini and consists of two clades, which associate with the type species of Premnobius and Premnophilus, and with morphological diagnostic characters. The following taxonomic changes are made based on the recovered relationships. Premnophilus is resurrected as a valid genus and Premnobiini is considered a sub-tribe of Ipini.

 Ipini systematics taxonomy nomenclature COI 16S 28S CAD Introduction

Premnobiini Browne (1962) consists of Premnobius Eichhoff (1879) and its 26 species that are endemic to sub-Saharan Africa; two of which are considered adventive to the Neotropics (Wood, 1982; Wood and Bright, 1992; Bright and Skidmore, 1997). Premnobius species are xylomycetophagus and are incestuous inbreeders, similar to Xyleborini (LeConte, 1876; Browne, 1961). This similarity in biology and gross morphology sparked debate on its taxonomic placement. Eichhoff (1878) originally suggested its affinity to Xyleborus Eichhoff (1864), however, subsequent researchers suggested alternative relationships with genera in the Corthylini, Dryocoeotini, and Xyloterini (Hagedorn, 1910; Hopkins, 1915). Schedl (1957) returned Premnobius to the Xyleborini and synonymized the genus with Xyleborus. Based on detailed examination of morphology and biology, Browne (1961) removed Premnobius from Xyleborus and described an additional closely related genus, Premnophilus Browne (1962). However, the tribal placement of Premnobius and Premnophilus remained uncertain despite the fact that Browne (1961) presented definitive morphological and behavioral evidence that these genera were unrelated to the Xyleborini. He suggested a new tribe, Premnobiini, for the genera and hypothesized its relationship between Ipini and Xyloterini. However, he postulated that these genera were Ipini, which converged to a xylomycetophagus habit (Browne, 1962). Premnophilus was soon synonymized with Premnobius because the morphological variation of its antennal club was considered within the range of variation observed for Xyleborus species (Schedl, 1964; Wood and Bright, 1992). Premnobiini was later formally established (Nobuchi, 1969), however, Wood (1982) did not recognize Premnobiini and placed Premnobius in Xyleborini stating that it represented the most primitive genus of the tribe. Phylogenetic studies including Premnobius cavipennis (Eichhoff, 1878) and various amount of molecular and morphological data demonstrated monophyly of Premnobius with Ipini genera (Normark et al., 1999; Farrell et al., 2001). Although no nomenclatural changes were made in these studies, subsequent researchers (Beaver, 2005; Bright and Torres, 2006; Alonso-Zarazaga and Lyal, 2009) recognized Premnobiini based on data from previous studies (Browne, 1961, 1962; Nobuchi, 1969; Normark et al., 1999). The most comprehensive phylogenetic analysis of scolytines placed Premnobius (two species) sister to Acanthotomicus and monophyletic with ten other Ipini species thus rendering Ipini paraphyletic (Jordal et al., 2011; Jordal and Cognato, 2012). The Premnobiini + Ipini clade was sister to the Xyleborini + Dryocoetini clade which both had 100% posterior probabilities and demonstrated, once again, the distant relation between Premnobiini and Xyleborini (Jordal and Cognato, 2012).

The previous phylogenetic studies did not address taxonomic or nomenclatural issues suggested by the resulting phylogenies (Normark et al., 1999; Farrell et al., 2001; Jordal et al., 2011; Jordal and Cognato, 2012). This study provides a more detailed phylogenetic analysis designed to address the tribal and generic status of Premnobiini and Premnophilus, respectively. Phylogenies reconstructed with molecular data for 79 species representing Dryocoetini (2 spp.), Premnobiini (15 spp.), Ipini (59 spp. including all genera), and Xyleborini (3 spp.) support the monophyly of Premnophilus and the inclusion of Premnobiini within Ipini. Taxonomic and nomenclatural changes concerning the Premnobiini are made based on these results.

 Materials and methods Taxa and DNA sequencing

DNA data for mtDNA cytochrome oxidase I (COI), mtDNA ribosomal 16S, nuclear ribosomal 28S (D2 and D3 regions), and nuclear protein coding gene CAD were generated for 22 species of Premnobiini and Acanthotomicus. The inclusion of existing data (Cognato and Sun, 2007; Jordal and Cognato, 2012) and the generation of CAD data created a data set of 79 species, which represented all Ipini genera and outgroup Dryocoetini and Xyleborini species (Table 1). Beetle specimens were collected over 20 years from various countries by AIC and his collaborators (Table 1). DNA was extracted from frozen and pinned specimens following the Qiagen (Hilden, Germany) tissue extraction protocol using Qiagen blood and tissue kits. Tissue was digested from the head and pronotum. These parts were recovered at the end of the extraction procedure. The head and pronotum and the remaining body were pinned and vouchered at the A.J. Cook Arthropod Research Collection, Michigan State University. Using the purified DNA, partial gene regions of COI, 16S, 28S, CAD were amplified using the primers and protocol listed in Table 2. Unincorporated nucleotides, primers and taq remaining in the PCR reactions were neutralized with ExoSAP-IT (USB Corp., Cleveland, OH) following the manufacturer protocols. Both strands of the clean PCR products were directly sequenced using BigDye Terminator v.1.1 (Applied Biosystems, Foster City, CA, USA) cycle sequencing kit and visualized on an ABI 3730 (Applied Biosystems) at the Research Technology Support Facility at Michigan State University. The DNA sequences were compiled and edited with Sequencher (GeneCodes, Ann Arbor, MI, USA) and archived at Genbank (Table 1).

Specimens included in current analyses with associated geographic localities and Genbank accession numbers.

Species DNA extraction codes Collection locations Genbank COI # Genbank 16S # Genbank 28S # Genebank CAD #
Acanthotomicus bicornatus Acabic Ghana: Bia KF862791 KF862713 KF862745 KF862827
Acanthotomicus fortis Aca.for1 Panama: BCI KF862792 KF862714 KF862746 KF862828
Acanthotomicus kepongi Akeg1 Thailand KF862793 KF862715 EU090306 KF862829
Acanthotomicus mimicus Aca.mim1 Panama: BCI KF862794 KF862716 KF862747 KF862830
Acanthotomicus sp. 1 SCI15A Sarawak KF862798 N/A KF862751 KF862834
Acanthotomicus sp. 2 AcaspB Ghana: Anakasa KF862796 N/A KF862749 KF862832
Acanthotomicus sp. 3 AcaspTH32 Thailand: Phu Khieo KF862797 KF862718 KF862750 KF862833
Acanthotomicus sp. 4 Pre.sp1 Cameroon: SW Province KF862806 KF862724 KF862760 KF862842
Acanthotomicus spinosus Acaspi Thailand: Nam Nao NP KF862795 KF862717 KF862748 KF862831
Ips acuminatus Ac1 Czech Republic: Moravia AF113325 KF862744 EU090296 KF862875
Ips avulsus Av5 USA: Louisiana AF113330 AF397472 EU090297 KF862876
Ips bonanseai Bon3 Mexico: Nuevo Leon AF113333 AF397473 EU090313 KF862877
Ips calligraphus Ca2 USA: New York AF113335 AF397475 EU090319 KF862878
Ips cembrae Ce3 Czech Republic: Moravia AF113337 AF397476 EU090320 KF862879
Ips duplicatus Du1 Czech Republic: Moravia AF113345 N/A KF862790 N/A
Ips emarginatus Em1 USA: California AF113347 AF397480 EU090325 KF862880
Ips grandicollis Gr2 USA: New York AF113349 AF397481 EU090323 KF862881
Ips hoppingi Ho5 USA: Arizona AF113354 AF397484 EU090321 KF862882
Ips knausi Knu1 USA: Colorado AF113357 AF397485 EU090327 N/A
Ips lecontei Le3 USA: Arizona AF113361 AF397486 EU090324 KF862883
Ips pilifrons Pil3 USA: Colorado AF113374 AF397493 EU090311 KF862884
Ips plastographus Pl4 USA: California AF113378 AF397492 N/A KF862885
Ips sexdentatus Sex2 Czech Republic: Moravia AF113380 AF397496 EU090314 N/A
Ips typographus Ty1 Czech Republic: Moravia AF113385 AF397499 EU090310 KF862886
Ips woodi Wo3 USA: California AF113389 AF397500 EU090322 KF862887
Orthotomicus caelatus Ort1 USA: New York AF113390 AF397501 EU090317 KF862868
Orthotomicus chaokhao Osp1 Thailand: Chiangmai KF862825 N/A KF862786 KF862870
Orthotomicus erosus Oer1 Greece: Athens U82236 AF397502 EU090302 KF862865
Orthotomicus proximus Opro Russia: St. Petersburg Region N/A N/A KF862784 N/A
Orthotomicus laricis Olar1 Romania: Retezat AF113392 N/A KF862783 N/A
Orthotomicus laricis Ort.gol1 Russia: Primorsky KF862823 KF862741 KF862782 KF862866
Orthotomicus latidens Lat3 USA: Washington AF113359 AF397503 EU090300 KF862872
Orthotomicus longicollis Olon1 Ukraine: Crimea N/A N/A EU090301 KF862867
Orthotomicus mannsfeldi Ma1 Austria: Vienna AF113363 AF397504 EU090312 KF862873
Orthotomicus nobilis Nob1 Canary Islands: Tenerife AF113366 AF397505 KF862788 N/A
Orthotomicus spinifer Sp1 USA: California AF113381 AF397506 KF862789 KF862874
Orthotomicus suturalis Osut1/Osut2 Russia KF862826 KF862743 KF862787 KF862871
Orthotomicus suturalis Osta1 China: Sichuan KF862824 KF862742 KF862785 KF862869
Pityogenes bidentatus P.bd1 Russia: St. Petersburg Region KF862815 KF862734 KF862776 KF862858
Pityogenes bistridentatus P.bs1 Ukraine: Crimea KF862816 KF862735 EU090304 KF862859
Pityogenes calcaratus PiCa2 Greece: Attica AF113394 N/A KF862777 KF862860
Pityogenes carinulatus Pito1 USA: New Mexico AF113393 N/A N/A KF862857
Pityogenes chalcographus P.ch1 Russia: St. Petersburg Region KF862814 N/A KF862775 N/A
Pityogenes fossifrons P.fos1 USA: Washington KF862817 KF862736 N/A KF862861
Pityogenes irkutensis Pir1 Russia: Moscow Region KF862818 KF862737 EU090305 KF862862
Pityogenes knechteli P.kne1 USA: Califorina KF862819 KF862738 KF862778 KF862863
Pityogenes plagiatus P.pla1 Canada: Ontario N/A N/A KF862780 N/A
Pityogenes porifrons Ppor1 Cyprus KF862821 N/A EU090307 KF862864
Pityogenes quadridens P.qua1 Russia: St. Petersburg Region KF862822 KF862740 KF862781 N/A
Pityokteines curvidens P.cur4 Crotia: Litoric N/A KF862730 KF862771 KF862854
Pityokteines lasiocarpi Pit.las1 USA: Washington KF862812 KF862731 KF862772 N/A
Pityokteines marketae P.mar2 Turkey: Mersin KF862820 KF862739 KF862779 N/A
Pityokteines minutus Pit.min1 USA: Oregon KF862813 KF862732 KF862773 KF862855
Pityokteines vorontzowi P.vor1 Crotia: Litoric EF534718 KF862733 KF862774 KF862856
Pityokteines sparsus Pk1 Canada: Quebec AF113396 N/A KF862770 KF862853
Premnobius adjunctus Preadj1 Cameroon: SW Province KF862799 N/A KF862752 KF862835
Premnobius ambitiosus Pre.amb1 Cameroon: SW Province KF862800 KF862719 KF862753 N/A
Premnobius cavipennis Precav1 Ghana: Bokuro-Abaa KF862801 N/A KF862754 KF862836
Premnobius cavipennis Precav3 Brazil: Bahia KF862802 KF862720 KF862755 KF862837
Premnobius corthyloides Precor1 Ghana: Bokuro-Abaa KF862803 KF862721 KF862756 KF862838
Premnobius orientalis Preori1 Tanzania: Udzungwa KF862805 N/A KF862758 KF862840
Premnobius quadrispinosus (Premnophilus) Pre.qua1 Cameroon: SW Province N/A KF862723 KF862759 KF862841
Premnobius sp. 2 (Premnophilus) PreX20 Ghana: Anakasa KF862809 N/A KF862766 KF862847
Premnobius sp. 3 PreX22 Ghana: Anakasa KF862810 KF862728 KF862767 KF862848
Premnobius sp. 4 Prex29 Ghana: Bia KF862811 KF862729 KF862768 KF862849
Premnobius sp. 5 (Premnophilus) Predor1 Ghana: Anakasa KF862804 KF862722 KF862757 KF862839
Premnobius sp. 6 (Premnophilus) Preunk1 Guyana: Iwokrama N/A N/A KF862764 KF862846
Premnobius sp. 7 (Premnophilus) PreX16 Ghana: Bokuro-Abaa KF862808 KF862727 KF862765 N/A
Premnobius sp. 8 Pre.sp2 Tanzania: Udzungwa N/A N/A KF862761 KF862843
Premnobius sp. 9 Pre.sp3 Madagascar: Ranomafara N/A KF862725 KF862762 KF862844
Premnobius sp. 10 Pre.sp4 Sierra Leone: Tiwai Isl. KF862807 KF862726 KF862763 KF862845
Pseudips concinnus CoCi3 USA: Washington AF113340 AF397511 EU090316 KF862850
Pseudips mexicanus Mx1 USA: California AF113364 AF397512 EU090299 KF862851
Pseudips orientalis Psori3 China: Qinghai GU811707 N/A KF862769 KF862852
OUTGROUPS
Dryocoetes autographus DrDry01 N/A JX263816 N/A HQ883565 HQ883791
Thamnurgus cylindricus DrCyr03 N/A JX263813 N/A JX263707 JX264025
Anisandrus dispar XyXyl02 N/A HQ883695 N/A HQ883606 HQ883840
Cnestus bimaculatus Cnebim N/A N/A N/A GU808579 GU808619
Xyleborus affinis Xylaff N/A N/A N/A GU808581 GU808621

N/A= not available do to PCR or sequencing issues.

PCR primers and cycling regimes.

Genes Primer names Primers References Cycling protocals
COI C1-J-2183 5′ -CAACATTTATTTTGATTTTTTGG-3′ Simon et al. (1994) 95°C (15 min); 40 cycles of 94°C (30 s)
C1-N-2611 5′ -GCAAAAACTGCACCTATTGA-3′ Simon et al. (1994) / 45°C (45 s) / 72°C (45 s); 72°C (5 min)
C1-J-2410 5′ -CCTACAGGAATTAAAATTTTTAGTTGATTAGC-3′ Simon et al. (1994) 95°C (15 min); 40 cycles of 94°C (30 s)
TL2-N-3014 5′ -TCCAATGCACTAATCTGCCATATTA-3′ Simon et al. (1994) / 52°C (45 s) / 72°C (45 s); 72°C (5 min)
16S LR-J-12961 5′ -TTTAATCCAACATCGAGG-3′ Cognato and Vogler (2001) 95°C (15 min); 40 cycles of 94°C (45 s)
LR-N-13398 5′ -CGCCTGTTTAACAAAAACAT-3′ Simon et al. (1994) / 50°C (45 s) / 72°C (45 s); 72°C (5 min)
28S D2F1 5′ -ACTGTTGGCGACGATGTTCT-3′ Jordal et al. (2008) 95°C (15 min); 40 cycles of 94°C (45 s)
D3R2 5′ -TCTTCGCCCCTATACCC-3′ Jordal et al. (2008) / 55°C (45 s) / 72°C (45 s); 72°C (5 min)
3665 5′ -AGACAGAGTTCAAGAGTACGTG-3′ Jordal et al. (2008)
4048 5′ -TTGCTCCGTGTTTCAAGACGGG-3′ Jordal et al. (2008)
CAD apCADforB2 5′ -TGGAARGARGTBGARTACGARGTGGYCG-3′ Danforth et al. (2006) 95°C (15 min); 40 cycles of 94°C (30 s)
apCADrevlmod 5′ -GCCATYRCTCBCCTACRCTYTTCAT-3′ Danforth et al. (2006) / 58°C (30 s) / 72°C (60 s); 72°C (5 min)
DNA sequence alignment and phylogenetic analyses

Protein coding genes were aligned without the aid of software computation given that insertion/deletions and introns were not observed. Ribosomal DNA sequences varied in their length. These sequences were manually aligned based on previous published alignments (Cognato and Vogler, 2001; Jordal et al., 2008) and using the alignment software, MUSCLE, with default parameters (Edgar, 2004). These data sets were treated separately in subsequent phylogenetic analyses.

Parsimony and maximum likelihood optimality criteria were used to reconstruct phylogenies. Parsimony analyses using PAUP* 4.0 b10 PPC (Swofford, 2002) consisted a heuristic search with 500 stepwise random addition replicates keeping only 500 suboptimal trees per replicate and the default settings with the manually and MUSCLE aligned data sets. These analyses were repeated while treating gap positions as 5th characters states. Bootstrap values were calculated with 500 pseudoreplicates for the four data treatments. Partition Bremer support was calculated with TreeRot v.2 (Sorenson, 1999). For the manually and MUSCLE aligned data sets, maximum likelihoods were estimated via Bayesian analysis using Mr. Bayes 3.2.2 (Ronquist et al., 2012). These analyses consisted of two simultaneous runs of four Metropolis-Coupled Markov chain Monte Carlo searches (one cold, three heated). Each search comprised 10 million generations which was sampled every 100th iteration. The data were partitioned by codon positions and ribosomal genes and each partition was allowed to independently evolve under a general time reversible (GTR + I + Γ) model. All parameters reached stability within 10 million generations and the split distribution between runs did not vary much (mean standard deviation between runs = 0.002). Bayesian posterior probabilities of clades were calculated by a majority-rule consensus of those trees after the burn-in (for both runs 75000 trees).

 Results and discussion Phylogenetic analyses

The combination of analyses and alignments resulted in tree topologies that differed by various sub-clades but were generally similar (e.g., Figures 1–3). For example, Ipini including Premnobiini was monophyletic and the clade consisting of Ips, Orthotomicus, Pityogenes, and Pityokteines was found in the majority of analyses (Table 3). Treating gapped positions as 5th character states in the parsimony analyses resulted in trees with many non-monophyletic genera. The monophyly of these genera is not suspect given the many morphological diagnostic and synapomorphic characters recognized for each genus (e.g., Hopping, 1963; Wood, 1986; Cognato, 2000). Given the lack of taxonomic congruence, the analyses that treated gapped positions as 5th character states were not given further consideration.

Phylogeny of Ipini and outgroup species; 1 of 87 most parsimonious trees based on the manually aligned data. Clades marked by “X” were not resolved in the strict consensus of the 87 mpts. Numbered clades are referenced in Table 4. Numbers = bootstrap values and clades without numbers had > 50% bootstrap values.

Phylogeny of Ipini and outgroup species; 1 of 756 most parsimonious trees based on the MUSCLE aligned data. Clades marked by “X” were not resolved in the strict consensus of the 756 mpts. Numbered clades are referenced in Table 5. Numbers = bootstrap values and clades without numbers had >50% bootstrap values.

Majority rule consensus of 75,000 trees found in a Bayesian analysis of Ipini and outgroup species. Numbers are posterior probabilities.

Summary of phylogenetic analyses.

Clades Parsimony Parsimony Bayesian Bayesian
Muscle alignment Manual alignment Muscle alignment Manual alignment
Gaps missing Gaps fifth state Gaps missing Gaps fifth state
Acanthotomicus NM NM NM NM NM NM
Ips 99 NM 99 NM 100 100
Orthotomicus 70 NM 57 NM 100 100
Pityogenes 100 78 99 NM 100 100
Pityokeines 98 NM 99 NM 100 100
Premnobius 100 NM 100 NM 100 100
Premnophilus 100 NM 89 NM 100 100
Pseudips 100 86 100 100 100 100
Ips + Orthotomicus >50 NM NM NM 100 NM
Pityokteines + O. longicollis 94 NM 93 NM 100 100
(Pityokteines + O. longicollis) P. lasicarpi) 56 NM >50 NM 99 100
Pityokteines + Pityogenes NM NM >50 NM NM 98
(Ips, Orthotomicus, Pityokteines, Pityogenes) 88 NM 94 NM 100 100
Pseudips + Acanthotomicus, in part NM NM NM NM 100 100
Premnobius + Premnophilus, i.e., Premnobiina 100 97 100 96 100 100
Premnobiina + Acanthotomicus, in part 59 68 61 93 100 96

Numbers, bootstrap or posterior probabilities; NM, not monophyletic in the strict consensus of mpts.

The alignments affected the distribution of branch support among the data partitions. For the manual alignment 16S exhibited two-three times more support than the other data sets (Table 4). Data interaction changed for the MUSCLE alignment. CAD and 28S exhibited two-six times more support than COI and 16S and support from 16S decreased by a third (Table 5). Also the MUSCLE alignment supported three times more clades as compared to the manual alignment (Tables 4 and 5). Changes in support given different alignments have been observed in other studies (e.g., Cognato and Vogler, 2001; Damgaard et al., 2004). An increase in support values was interpreted as increased concordance of data interaction (Cognato and Vogler, 2001). Thus, the parsimony and Bayesian phylogenies (Figures 2, 3) based on the MUSCLE aligned data with gap positions scored as missing were considered as better hypotheses as compared to the trees found with the manually aligned data.

Partition bremer support for 1 of 87 most parsimonius trees reconstructed with the manually aligned data (Figure 1).

Node COI 16s 28s CAD Total
1 0 0 0 0 0
2 0 2.5 −2 −0.5 0
3 0 2.5 −2 −0.5 0
4 0 0 0 0 0
5 0 0 0 0 0
6 0 2.5 −2 −0.5 0
7 0 2.5 −2 −0.5 0
8 0 0 0 0 0
9 0 2.5 −2 −0.5 0
10 0 −2.5 2 0.5 0
11 0 0 0 0 0
12 0 2.5 −2 −0.5 0
13 0 0 0 0 0
14 0 0 0 0 0
15 1 −0.5 20 18.5 39
16 8 1.5 8 2.5 20
17 0 2.5 −2 −0.5 0
18 0 0 0 0 0
19 0 −2.5 2 0.5 0
20 0 0 0 0 0
21 0 4 8 0 12
22 0 2.5 −2 −0.5 0
23 0 0 0 0 0
24 0 0 0 0 0
25 0 0 0 0 0
26 0 2.5 −2 −0.5 0
27 0 0 0 0 0
28 0 0 0 0 0
29 0 2.5 −2 −0.5 0
30 0 0 0 0 0
31 0 0 0 0 0
32 0 0 0 0 0
33 0 0 0 0 0
34 0 −2.5 2 0.5 0
35 0 0 0 0 0
36 0 −0.5 −1 2.5 1
37 0 −2.5 2 0.5 0
38 0 −0.8 0.7 0.2 0
39 0 −0.5 −1 2.5 1
40 0 0 0 0 0
41 0 2.5 −2 −0.5 0
42 0 0 0 0 0
43 0 0 0 0 0
44 −12.5 27 −1 −6.5 7
45 0 0 0 0 0
46 0 0 0 0 0
47 0 0 0 0 0
48 0 0 0 0 0
49 0 0 0 0 0
50 0 0 0 0 0
51 0 2.5 −2 −0.5 0
52 0 0 0 0 0
53 0 0 0 0 0
54 0 0 0 0 0
55 −6 8 7 −4 5
56 −6 8 7 −4 5
57 −4 5.5 3.5 8 13
58 0 0 0 0 0
59 0 0 0 0 0
60 0 2.5 −2 −0.5 0
61 0 0 0 0 0
62 26 16.5 −1 −3.5 38
63 −2 4.5 −1 −0.5 1
64 0 2.5 −2 −0.5 0
65 0 0 0 0 0
66 0 0 0 0 0
67 0 0 0 0 0
68 11 3 −0.5 8.5 22
69 12.5 20 −2 5.5 36
70 0 0 0 0 0
71 9 33.5 30 −0.5 72
72 9 11.5 12 5.5 38
73 14.5 1.5 −33.5 41.5 24
74 1 2.5 8 13.5 25
75 0 2.5 −3 3.5 3
76 −7.2 −1.2 31.5 −1 22
Total 54.3 171 71.7 87.2

Partition bremer support for 1 of 756 most parsimonius trees reconstructed with the MUSCLE aligned data (Figure 2).

Node COI 16s 28s CAD Total
1 0 0.3 5.6 3.1 9
2 −0.2 0.4 −1.2 4.1 3
3 0 0.3 −1.4 1.1 0
4 0 0.1 −1.4 1.4 0
5 0 0.1 −1.4 1.4 0
6 0 0.3 −1.3 1.1 0
7 0 0.3 −1.3 1.1 0
8 0 0.4 −1.5 1.2 0
9 0 0.3 1.5 2.2 4
10 0 0.3 3.5 10.1 14
11 0 0 0 2 2
12 −0.1 4.1 −1.4 −0.6 2
13 0 4.1 −1.4 −0.6 2
14 −2.1 −0.4 −0.5 11 8
15 25.4 −10.7 18.6 0.6 34
16 −2.2 −0.3 −2.5 6 1
17 −2.6 0.6 3.8 4.2 6
18 −20.9 −0.2 9.9 16.3 5
19 1.8 −0.6 −6.6 10.4 5
20 −22.6 0.6 8.6 18.4 5
21 −0.3 0.3 −1.1 3.1 2
22 −0.1 0.3 1.8 1 3
23 −0.3 0.3 6.6 3.4 10
24 −0.6 0.6 −5.4 20.4 15
25 0.7 0.1 8 0.3 9
26 −0.2 2 2.6 2.6 7
27 −0.1 0.3 1.7 1.1 3
28 −0.3 0.4 4.8 1.1 6
29 −1.6 −1.1 7 0.7 5
30 0.4 0.3 11.8 0.5 13
31 16.8 0.1 −1.4 0.4 16
32 8.4 −1.4 6.6 −3.6 10
33 18.4 3.6 7.6 4.4 34
34 2.9 1.7 4.4 5 14
35 10.4 1.6 2.6 17.4 32
36 −4.6 4.6 7.6 −2.6 5
37 −0.3 −1.3 7.5 −4.9 1
38 0.4 0.6 −3.4 2.4 0
39 −0.4 0.6 −1.6 3.4 2
40 2.4 0.6 −3.4 2.3 2
41 −2.7 3.8 −1.2 4.1 4
42 −0.6 0.6 −3.4 3.4 0
43 −1.6 0.9 −2 2.7 0
44 −1.5 1 −2.3 2.8 0
45 −1 1.4 0.1 1.5 2
46 −0.8 0.8 −2.6 2.6 0
47 −0.1 3.1 1.6 2.4 7
48 −2.1 −6.4 0.1 11.4 3
49 −2.2 3.7 8.2 −0.7 9
50 26.9 5.6 2.1 3.4 38
51 2.9 3.1 −2.3 1.3 5
52 5.1 7.2 −1.4 7.1 18
53 −3.3 1.6 5.6 0 4
54 3.4 −1.4 −8.4 8.4 2
55 0 0 0.1 −0.1 0
56 −2.6 −0.4 8.2 −2.2 3
57 −2.6 −0.4 5.1 −2.1 0
58 −2.7 −0.4 5.1 −2.1 0
59 −0.6 −0.4 2.6 0.4 2
60 0.3 0.4 9.3 1.1 11
61 −1.8 −0.1 2.5 7.4 8
62 −0.2 0.3 3.7 1.1 5
63 −2.6 −0.4 14.1 −2.1 9
64 −2.5 −0.5 19.2 4.8 21
65 −4.6 3.6 8.6 5.4 13
66 −2.7 −0.3 5.1 −2.1 0
67 −1.6 −0.4 −3.9 6.8 1
68 1.4 4.6 6.1 7.9 20
69 14.9 6.6 −2.6 5.1 24
70 −2.5 −0.5 5.2 −2.3 0
71 6.8 0.4 −0.2 4 11
72 1.8 0.4 3 21.9 27
73 −1.3 −0.5 8.4 15.4 22
74 −2.1 −0.4 1.1 11.4 10
75 −0.3 0.2 −0.5 4.6 4
76 −0.4 0.2 −4.2 10.4 6
Total 45 51.2 184 298

Both analyses mostly recovered consistent results with all genera (except Acanthotomicus) and Premnobiina as monophyletic. However, the relationships of these genera differed between the analyses. Acanthotomicus, in part, or Pseudips was sister to the other Ipini genera, however, there was no to little support for this position of the latter genus (Figure 1). The position of Pseudips imbedded within Acathotomicus agreed with previous phylogenies that demonstrated that Pseudips was not monophyletic with Ips (Cognato, 2000) or other Holarctic Ipini genera (Jordal and Cognato, 2012). This result also supported the validity of Pseudips as distinct from Orthotomicus (Wood, 2007; Alonso-Zarazaga and Lyal, 2009; Knížek, 2011).

Acanthotomicus was polyphyletic (Figures 1–3) and confirmed similar results of a study which included fewer Acanthotomicus specimens (Jordal and Cognato, 2012). Clades did not associate with geographic region because several clades comprised a mixture of species from Asia, Africa and/or South America (Figures 1–3). This was not expected because the pantropical distribution of the genus predicts that genetic isolation would associate with geographic distance (Avise, 2000). Although there is a gestalt to their appearance which suggests monophyly of the genus, groups of species differ in details of antennal and elytral morphology. For example, a pattern of morphological concordance was present, for a clade including A. spinosus, A. sp. 1/Sarawak, and A. sp. 3/Thailand (Figure 3) which have long spines on their elytral declivity. Additional patterns may emerge with the inclusion of the ~90% remaining Acanthotomicus species in future phylogenetic analyses which will provide better hypotheses of interspecific and intergeneric relationships of Acanthotomicus. However, it appears unlikely that a monophyletic Acanthotomicus will be resolved, given the high support for these phylogenies (Figure 3) and others (Jordal and Cognato, 2012) thus a thorough revision of the genus will be necessary.

The remaining Holarctic genera, Ips, Orthotomicus, Pityogenes, and Pityokteines, were monophyletic, except for one Orthotomicus species included in Pityokteines, but the relationships among the genera varied and were not well-supported (Table 3, Figures 1, 2). Details of the species relationships for each genus will be the subject of future studies. However, two conspicuous results will be discussed. First, O. longicollis (Gyllenhal, 1827) was monophyletic with Pityokteines (Figures 1–3) as previously observed within a 28S based phylogeny (Jordal et al., 2008). This species has an obliquely truncated antennal club with inconspicuous sutures similar to the antennal club of Pityokteines. The generic placement of O. longicollis in Pityokteines appears appropriate but additional nucleotide data is needed to confirm this relationship. Second, Pityokteines lasiocarpi (Swaine, 1916), which was once considered a monotypic genus, Orthotomides Wood (1951), was sister to the Pityokteines species including O. longicollis (Figures 1–3). Although the structure of the antennae of P. lasisocarpi is not typical of Pityokteines, there is currently little reason for the resurrection of Orthotomides.

The clade including Premnobius and Premnophilus was well-supported (Table 3, Figures 1–3). This clade was sister to either of two African Acanthotomicus species and well-imbedded amongst other Ipini genera. Given this phylogenetic position, the tribal status of this clade is unjustified; see below. Premnophilus was reciprocally monophyletic with Premnobius and included P. quadrispinosus (Schedl, 1938) and three unidentified species (likely undescribed). Given a well-supported clade and morphological diagnostic characters, Premnophilus is recognized as distinct from Premnobius; see below. Premnobius was also well-supported (Figures 1–3) and include species that were characterized by a carinate lateral eytral margin. These species resembled P. cavipennis and differed by minor morphological differences of the elytral declivity (clade 9, Figure 2). One species rendered P. cavipennis paraphyletic, which suggests the need of a revision. Terminal branch lengths of unidentified species were similar in length to terminal branch lengths of known species (e.g., P. sp. 3/Ghana and P. ambitiosus) (Figure 3). If branch lengths along with minor morphological differences are taken as a proxy for species delimitation, then a greater Premnobius species diversity awaits discovery.

 Taxonomy <italic>Premnophilus</italic> browne, <italic>genus bona</italic> (Figures <xref ref-type="fig" rid="F4">4</xref>, <xref ref-type="fig" rid="F5">5</xref>)

Premnophilus (Browne, 1962): 79. Type species: Xyleborus joveri (Schedl, 1951)= Premnobius quadrispinosus (Schedl, 1938); original designation.

Lateral view of the holotype of Premnophilus quadrispinosus, the type species of Premnophilus. Specimen length is approximately 1.5 mm.

Antenna of Premnophilus sp.

 Diagnosis

Browne (1962) thoroughly described and illustrated the genus. To emphasize, Premnophilus differs from Premnobius by the obliquely truncated antennal club without obvious sutures. The anterior edge of the first corneous segment is sinuous. In Premnobius the antennal club is flat with two procurved sutures and the anterior edge of the first corneous segment is procurved. Also, the lateral edge of the eytral declivity for Premnophilus is rounded as compared to the acute lateral edge of the eytral declivity for Premnobius.

 Included species

I have observed specimens of the listed species. Two or three likely undescribed species (e.g., P. sp. 5/Ghana, P. sp. 6/Guyana, Figure 1) also belong to Premnophilus. A revision of this genus will occur in a subsequent publication (Cognato, in prep.). Complete references are given in Wood and Bright (1992).

 <italic>Premnophilus quadrispinosus</italic> (schedl), new combination

Premnobius quadrispinosus Schedl, 1938: 461.

Xyleborus joveri Schedl, 1951: 41–42.

 <italic>Premnophilus perspinidens</italic> (schedl), new combination

Xyleborus perspinidens Schedl, 1957: 107.

 Premnobiina, <italic>status novo</italic>

Included genera.

Premnobius Eichhoff, 1879.

Premnophilus Browne, 1962.

As indicated in the results, Premnobius and Premnophilus are monophyletic and sister to Acanthotomicus, in part (Figures 1–3). Consideration of these genera as a tribe renders Ipini paraphyletic. To remedy this issue, Premnobiini is recognized as a sub-tribe of Ipini. Alternatively, clades of Acanthotomicus and Pseudips could be recognized as tribes to preserve the monophyly of Ipini and the tribal status of Premnobiini. However, this action is not advisable because of the limited sample of Acanthotomicus species included in these analyses.

 Conflict of interest statement

The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

I thank Sarah M. Smith, Bjarte H. Jordal, and the Review Editors for helpful discussion of this topic and for review of previous manuscripts. I appreciate the collecting efforts of Jiri Hulcr, Bjarte H. Jordal, and Sarah M. Smith, which helped make this study possible. This study was supported in part by the Norwegian Research Council (214232) and the University of Bergen, Norway (F20) awarded to Bjarte H. Jordal. and NSF-PEET (DEB 0328920) awarded to Anthony I. Cognato.

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