Molecular Phylogenetics and Evolution 64 (2012) 428–440
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Molecular Phylogenetics and Evolution
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Molecular phylogenetics of a South Pacific sap beetle species complex (Carpophilus spp., Coleoptera: Nitidulidae) ⇑ Samuel D.J. Brown a, , Karen F. Armstrong a, Robert H. Cruickshank b a Bio-Protection Research Centre, PO Box 84, Lincoln University, Lincoln 7647, Canterbury, New Zealand b Department of Agriculture and Life Sciences, PO Box 84, Lincoln University, Lincoln 7647, Canterbury, New Zealand article info abstract
Article history: Several species of sap beetles in the genus Carpophilus are minor pests of fresh produce and stored prod- Received 19 May 2011 ucts, and are frequently intercepted in biosecurity operations. In the South Pacific region, the superficially Revised 28 March 2012 similar species C. maculatus and C. oculatus are frequently encountered in these situations. Three subspe- Accepted 30 April 2012 cies of C. oculatus have been described, and the complex of these four taxa has led to inaccurate identi- Available online 18 May 2012 fication and questions regarding the validity of these taxa. A molecular phylogenetic study using the mitochondrial gene cytochrome c oxidase I (COI) and two nuclear markers comprising the rDNA internal Keywords: transcribed spacer 2 (ITS2) and the D1–D2 region of the large (28S) ribosomal RNA subunit showed that C. Oceania maculatus, and C. o. cheesmani were easily differentiated from the two other subspecies of C. oculatus. COI Polynesia Taxonomy also showed differentiation between C. o. gilloglyi and C. o. oculatus, but this was not shown when third biosecurity codon positions were removed and when RY-coding analyses were conducted. Generalised mixed Yule- Coalescent (GMYC) models were fitted to trees estimated from the COI data and were analysed using a multimodel approach to consider the evidence for three taxonomic groupings of the C. oculatus group. While the arrangement with the highest cumulative weight was not the arrangement ultimately accepted, the accepted taxonomy also had an acceptable level of support. ITS2 showed structure within C. oculatus, however C. o. oculatus was resolved as paraphyletic with respect to C. o. gilloglyi. COI showed evidence of sequence saturation and did not adequately resolve higher relationships between species represented in the dataset. 28S resolved higher relationships, but did not perform well at the species level. This study supports the validity of C. maculatus as a separate species, and provides sufficient evi- dence to raise C. o. cheesmani to the level of species. This study also shows significant structure within and between C. o. gilloglyi and C. o. oculatus, giving an indication of recent speciation events occurring. To highlight the interesting biology between these two taxa, C. o. gilloglyi is retained as a subspecies of C. oculatus. These results give clarity regarding the taxonomic status of C. maculatus and the subspecies of C. oculatus and provide a platform for future systematic research on Carpophilus. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction disease (Cease and Juzwik, 2001). The species most commonly found in stored products are C. hemipterus, C. dimidiatus (Fabricius), The sap beetle genus Carpophilus (Coleoptera: Nitidulidae) C. ligneus Murray and C. obsoletus Erichson (Dobson, 1955). A num- contains over 200 species found throughout the world. Both adults ber of species are also associated with flowers and are important and larvae live and develop in organic matter, including rotting pollinators, particularly of the Annonaceae (Nagel et al., 1989). In fruit and stored products, which has made some species pests in the tropical South Pacific region, C. maculatus Murray and C. ocula- orchards and warehouses. Carpophilus davidsoni Dobson, C. hemi- tus Murray are widespread species that are commonly found in pterus (Linnaeus) and C. mutilatus Erichson have emerged as seri- agricultural situations, and have been intercepted in exported fresh ous pests of stone fruit in Australia with the decrease in use of produce (Archibald and Chalmers, 1983; Leschen and Marris, broad-spectrum insecticides (Hossain and Williams, 2003; James 2005). et al., 1997; James and Vogele, 2000), while C. lugubris Murray is There has been much published on the ecology and control of an important pest of corn in the United States (Dowd, 2000) and the group, as it is considered to be the most economically impor- C. sayi Parsons has been implicated in the transmission of oak wilt tant genus within the Nitidulidae (Connell, 1981). However, the taxonomy of the genus remains problematic, with the last global ⇑ Corresponding author. revision of the group dating to the 19th Century (Murray, 1864). E-mail address: [email protected] (S.D.J. Brown). Since Murray’s seminal work, several new species have been
1055-7903/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ympev.2012.04.018 S.D.J. Brown et al. / Molecular Phylogenetics and Evolution 64 (2012) 428–440 429 described, most notably by Dobson and Kirejtshuk (e.g. Dobson, 2. Methods 1952, 1993a; Kirejtschuk, 2001). Not withstanding this, the genus remains in need of a thorough taxonomic revision, as most species 2.1. Taxon coverage and specimens are poorly defined and morphologically variable, with subtle char- acters defining the species. This taxonomic uncertainty impacts on To test the monophyly of C. oculatus, the three subspecies of this the ability to understand their relative invasiveness and risk. species were collected preferentially. The relationship of C. oculatus Within the Nitidulidae, Carpophilus is placed in the subfamily with C. maculatus was also targeted, given the morphological sim- Carpophilinae (Kirejtschuk, 2008). While no formal systematic ilarity between them. Beyond this, other Carpophilus species were studies have yet been published on the relationships of higher taxa collected opportunistically by the senior author and colleagues, within the Nitidulidae, it is believed that the Carpophilinae form a primarily from the Pacific. single lineage with the Epuraeinae and Amphicrossinae (Kirejts- Specimens were collected by hand from rotting fruit and vege- chuk, 2008), an arrangement that is consistent with preliminary tables and preserved in propylene glycol in the field for transport- molecular systematic results from the family (A. Cline pers. comm.). ing back to New Zealand. In the laboratory, specimens were sorted The genus is easily identified by having two exposed abdominal and temporarily stored in 100% ethanol. A single leg was removed tergites and a button-like male 8th sternite (Gillogly, 1962; Leschen from each specimen to be used for molecular analysis and the and Marris, 2005). It is morphologically conservative between remainder card-mounted as a voucher specimen and deposited in species within the genus and there are a number of groups contain- the Lincoln University Entomology Museum (LUNZ). ing several species with very similar appearance and habits. Carpo- philus is classified into nine subgenera: Carpophilus Stephens sensu 2.2. Molecular methods stricto, Megacarpolus Reitter, Semocarpolus Kirejtshuk, Gaplocarpo- lus Kirejtshuk, Askocarpolus Kirejtshuk, Plapennipolus Kirejtshuk, DNA was extracted using the prepGEM DNA extraction kit (Zy- Ecnomorphus Motschulsky, Caplothorax Kirejtshuk and Myothorax GEM Ltd., Hamilton, New Zealand). Incubation consisted of 30 min Murray (Kirejtschuk, 2008). However, the exact limits of these sub- at 75 °C and 5 min at 95 °C. This longer incubation period ensured genera have not been expanded on in the context of an overall re- that dehydrated tissues had sufficient time to rehydrate and lyse. view of Carpophilus systematics. The majority of these subgenera DNA was amplified using a 10 ll PCR reaction containing 0.25 U are fairly small, consisting of only a few species, most of which Expand High-Fidelity Taq (Roche Applied Science, Indianapolis, are confined to South-East Asia. The exceptions are Carpophilus s. IN, USA), 0.2 mM dNTPs, 2 mM MgCl and 0.3 mM of both forward str., Ecnomorphus and Myothorax, which are large and cosmopolitan. 2 and reverse primers. To encourage DNA amplification in difficult Carpophilus maculatus and C. oculatus are widely distributed specimens, 2 ll GC Rich mixture (Roche) was added to the reaction through the South Pacific, with C. maculatus also being found in when necessary. The 50 end of the cytochrome c oxidase subunit I Southeast Asia, South America and the West Indies. The two spe- (COI) mitochondrial gene, the D1–D2 region of the 28S ribosomal cies are similar, with the key character that differentiates between RNA gene and the internal transcribed spacer 2 (ITS2) region in them being the extent of red colouration on the elytra. However, the nuclear ribosomal encoding cistrons were amplified using the this patterning is very variable and there is significant overlap in primers listed in Table 1. For COI the combination LCO1490/ the patterns possessed by both species. Dobson (1993b) reviewed HCO2198 was used preferentially, with TY-J-1460/C1-N-2191 used C. oculatus and described three subspecies based on differences when these amplifications were unsuccessful. ITS2 amplifications in pronotal punctation and male genitalia. The nominate subspe- required a primer concentration of 0.5 mM. Reactions were run cies, C. o. oculatus is found from Tahiti through the central Pacific on a GeneAmp 9700 thermocycler (Applied Biosystems, Foster City, to New Caledonia. There is significant overlap in geographic range CA, USA) with an initial denaturation of 94 °C for 2 min, followed between this subspecies and C. o. gilloglyi Dobson which is found by 40 cycles of 94 °C (15 s), 45 °C (30 s) and 72 °C (75 s), and with from Easter Island to Fiji and Micronesia. The last subspecies, C. a final extension at 72 °C for 7 min. An annealing temperature of o. cheesmani Dobson is confined to Vanuatu. Morphological 54 °C was used for 28S reactions. Successful amplification was con- evidence for monophyly of the C. oculatus subspecies include the firmed by running PCR products in 1.0% agarose gels made using a distinctive ring-shaped elytral colour pattern and the pronotal NaOH/borate buffer with a pH of 8.0; these were run at 170 V and length/width ratio, however there are no clear and unambiguous 50 mA for 15 min. Gels were stained during casting with SYBR Safe synapomorphies that define C. oculatus. A sister taxon relationship DNA gel stain (Invitrogen, Carlsbad, CA, USA) and viewed with a is suggested between C. oculatus and C. maculatus by the sculptur- GeneWizard Gel imaging system (SynGene, Cambridge, England). ing of the prosternum and by the shape of the male genitalia. The PCR products were sequenced in 10 ll reactions containing 0.3 ll genitalic similarity is particularly striking between C. maculatus of PCR product, 0.5 ll BigDyeÒ Terminator (v3.1) (Applied Biosys- and C. o. gilloglyi, with the shape of the parameres of these two spe- tems), 2 ll BigDyeÒ Sequencing buffer (v1.1/3.1) and 0.8 lMof cies being nearly identical. These similarities, and the variation in the primers used for amplification. Products were sequenced in colour pattern has led some to surmise that these two species form both directions. PCR cleanup was done with CleanSEQÒ Dye- a single, hyper-variable species (Leschen and Marris, 2005). Terminator Removal kit (Agencourt Bioscience Corporation, In this study, the monophyly of C. oculatus and its relationship Beverly, MA, USA). Sequences were read using a Long Read with C. maculatus was tested using molecular systematic tech- Sequencing Protocol on an ABI PrismÒ 3100-Avant Genetic niques. The opportunity was also taken to provide a preliminary Analyzer (Applied Biosystems). glimpse into the systematics of Carpophilus, by sampling a range of species within the genus and with an emphasis on species with- in the subgenus Myothorax. A molecular phylogeny of Carpophilus 2.3. Analysis was constructed based on sequences of mitochondrial and nuclear gene regions from as many species as it was possible to obtain. This Sequences were aligned by eye using the manual alignment was used primarily to test the monophyly of C. oculatus and its software BIOEDIT (Hall, 1999). COI data were analysed using four subspecies, and to test for a sister-taxon relationship between methods, (1) Original nucleotide (NT) coded data, (2) Third codon C. oculatus and C. maculatus. It also provides a basis for the system- positions excluded, (3) Third codon positions RY-coded, and (4) atics of Carpophilus in general and will be useful for guiding future All data RY-coded (Phillips et al., 2004), while the other two loci taxonomic work on the genus. were unaltered. Loci were analysed independently, due to the 430 S.D.J. Brown et al. / Molecular Phylogenetics and Evolution 64 (2012) 428–440
Table 1 Markers and PCR primer combinations used in this research.
Marker Primer name Primer sequence Reference COI LCO1490 50-GGT CAA CAA ATC ATA AAG ATA TTG G-30 Folmer et al. (1994) HCO2198 50-TAA ACT TCA GGG TGA CCA AAA AAT CA-30 Folmer et al. (1994) TY-J-1460 50-TAC AAT TTA TCG CCT AAA CTT CAG CC-30 Simon et al. (1994) C1-N-2191 50-CCC GGT AAA ATT AAA ATA TAA ACT TC-30 Simon et al. (1994) 28S LSUfw2 50-ACA AGT ACC DTR AGG GAA AGT TG-30 Sonnenberg et al. (2007) LSUrev1 50-TAC TAG AAG GTT CGA TTA GTC-30 Sonnenberg et al. (2007) ITS2 CAS5p8sFc 50-TGAA CAT CGA CAT TTY GAA CGC ACA T-30 Ji et al. (2003) CAS28sB1d 50-TTC TTT TCC TCC SCT TAY TRA TAT GCT TAA-30 Ji et al. (2003)
inconsistencies in phylogenetic inference when genes with differ- (U. humeralis (Fabricius)) was also included in the dataset. COI and ing histories are concatenated (Kubatko and Degnan, 2007), and 28S were sequenced from all species, with the exception of C. the lack of multiple samples for many species precluded the use bakewelli and C. obsoletus for which 28S was unable to be amplified. of coalescent-based approaches (Heled and Drummond, 2010). ITS2 was only sequenced from selected species within the Myotho- Maximum likelihood (ML) analyses for all loci were run using rax subgenus. Outgroups were selected from available specimens PHYML ALRT (Anisimova and Gascuel, 2006; Guindon and Gascuel, and previously published sequences of other species within the 2003), and support for the topology calculated using parametric Nitidulidae. These represented three subfamilies outside of the bootstrap procedures with 1000 replicates. JMODELTEST (Posada, Carpophilinae and included Epuraea signata Broun and E. ocularis 2008) was used to determine the appropriate models of molecular Fairmaire (Epuraeinae), unidentified Conotelus and Brachypeplus evolution, based on the Akaike information criterion (AIC). Satura- species (Cillaeinae), and Aethina concolor (Macleay), Omosita discoi- tion plots of the p-distance against ML distances calculated from dea (Fabricius), three Meligethes species and unidentified specimens trees, were constructed in R using functions from the APE package of Phenolia and Stelidota (Nitidulinae). (Paradis et al., 2004; R Development Core Team, 2012). Pairwise distances are reported as both raw p-distances and as model-cor- 3.2. Alignments and variation rected distances to illustrate the uncertainty in the derivation of genetic distance estimates (Collins et al., in press). A total of 200 sequences over the three markers were obtained. Ultrametric COI trees for generalised mixed Yule-Coalescent Sequences have been submitted to Genbank with accession num- (GMYC) analysis were estimated from the NT coded COI data using bers GU217433–GU217530 for COI, GU217391–GU217432 for BEAST 1.6.2 (Drummond and Rambaut, 2007; Drummond et al., 28S and GU217338–GU217390 for ITS2. Specimen details are 2006) using an HKY + I + C model of evolution, a relaxed lognormal available as Supplementary Material (Table 6). Sequence polymor- molecular clock and default priors. A lognormal molecular clock phism data for each gene are presented in Table 2. model was justified with the ucld.stdev value of 1.384 indicating 28S and ITS2 alignments required the addition of alignment considerable rate heterogeneity. The MCMC chain was run for a to- gaps because of the presence of indels. While there are no indels tal of 50.7 million generations, and trees were sampled every 1000 in the COI dataset, 11 specimens had missing values from short generations. A maximum clade credibility (MCC) tree was made sequencing runs. The maximum number of missing bases in any using TREEANNOTATOR from the sampled trees after removing a stan- one specimen was 141, with the median number being 9. Base fre- dard burn-in of the first 10% trees of each run. GMYC (Pons et al., quencies of the three markers are typical for insects (Lin and Dan- 2006) was conducted on the MCC tree, and on 2,000 other ran- forth, 2004). domly selected trees generated by BEAST. The number and compo- Although COI and 28S had roughly equivalent levels of variable sition of clusters estimated using both single and multiple GMYC sites (Table 2), COI had more parsimony-informative sites than 28S, threshold were calculated. The composition of clusters, AIC, AIC and was better at distinguishing between species and subspecies of weights, model-weighted number of entities (Powell, 2012) and Carpophilus (Fig. 2 c.f. Fig. 3). Distribution of parsimony-informative other statistics on GMYC objects were calculated using SPIDER V. sites in COI across first, second and third codon positions respec- 1.1-2 and SPIDERDEV V. 0.0-3 (Brown et al., 2012). tively was 37, 5, and 159, a result consistent with most protein- To investigate the probability of trees based on a priori expecta- coding genes (Xia, 1998). The saturation plot (Fig. 1) for COI showed tions (i.e. monophyly of C. oculatus and Carpophilus), SH-tests (Shi- deviation from linearity, suggesting that the marker is unsuitable modaira and Hasegawa, 1999) were conducted using the SH.test for inferring higher relationships. Both nuclear markers are function in the PHANGORN package for R (Schliep, 2011).
Table 2 3. Results Sequence polymorphism data.
3.1. Taxon sampling COI 28S ITS2 Sequences 104 43 53 Specimens of 17 species of Carpophilus were sequenced. These Species 29 22 6 Length 576 734 504 represented four subgenera, as proposed by Kirejtschuk (2008). Gaps 0 135 169 Semocarpolus was represented by a single species, C. marginellus; Variable sites (%) 219 (38) 226 (38) 49 (15) Carpophilus by three species (C. lugubris, C. obsoletus and C. hemipte- Parsimony informative sites 201 133 27 rus); Ecnomorphus by four (C. antiquus Melsheimer, C. discoideus Base frequencies Leconte, C. bakewelli Murray and C. corticinus Erichson); and A 28.0 20.8 24.9 Myothorax by eight species (C. dimidiatus, C. mutilatus, C. davidsoni, C 19.6 25.5 20.2 C. gaveni Dobson, C. nepos Murray, C. maculatus, C. oculatus, C. robu- G 16.9 31.5 23.2 T 35.5 22.2 31.7 stus Murray and C. schiodtei Murray). A single species of Urophorus S.D.J. Brown et al. / Molecular Phylogenetics and Evolution 64 (2012) 428–440 431
COI ITS2 28S p−distance p−distance p−distance 0.0 0.10 0.15 0.20 0.00 0.05 0.10 0.15 0.20 0.00 5 0.00 0.05 0.10 0.15 0.20 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 ML distance ML distance ML distance
Fig. 1. Saturation plots of p-distances against ML distances based on the optimal model. The solid line shows the 1:1 relationship between p-distances and ML distances. essentially linear, showing no evidence of saturation at the level retained throughout the modifications to the dataset, though the investigated here. exact relationships vary between the modifications (see Supplementary Material). When third codons are removed, the 3.3. Phylogenetic analyses Fijian clade of C. o. gilloglyi is nested within C. o. oculatus, while the eastern clade is paraphyletic to the clade formed by the former 3.3.1. COI groups. These relationships, however, do not have bootstrap sup- 3.3.1.1. Maximum likelihood analyses. In all four COI ML analyses, C. port, while the monophyly of C. o. cheesmani is supported with a o. gilloglyi + C. o. oculatus are shown to form a monophyletic clade. bootstrap of 74.3%. In both RY-coded datasets, the Fijian clade is This clade is supported by >50% bootstrap values in all analyses, sister to the Eastern clade + C. o. oculatus (see Supplementary with the exception of the dataset where third codon positions were Material). excluded. None of the analyses resolved the sister-group relation- A deep split is also seen between Australian and Pacific popula- ship of C. o. cheesmani with statistical support or placed them with tions of C. maculatus, with a mean ML pairwise distance between the other two C. oculatus subspecies. The full NT-coded dataset the clades of 14.52% (6.01% mean p-distance). There was also sig- resolves C. o. gilloglyi and C. o. oculatus as being reciprocally mono- nificant structuring within the Pacific clade, with a mean ML pair- phyletic, while all other datasets resolve C. o. gilloglyi as paraphy- wise distance of 4.67% (1.9% mean p-distance). Within a single, letic with respect to C. o. oculatus. small island (Wallis), a specimen was 3.87% ML distance (2.62% Maximum likelihood of the nucleotide-coded COI data inferred p-distance) different from its conspecifics from the same island. a single tree of -ln likelihood 8179.84071 from a TPM3uf + I + C Within other species with a sample size that allows the calcula- model with nucleotide frequencies and rate parameters as shown tion of intra-specific distances, C. nepos had a mean ML pairwise in Tables 2 and 3. The tree (Fig. 2) grouped conspecific taxa with distance of 4.18% (1.59% mean p-distance) from three specimens each other, and were supported with high bootstrap values. In collected in New Caledonia, Tahiti and a laboratory culture from particular, the monophyly of C. o. oculatus and C. o. gilloglyi is the USA. In ML analyses, the specimen from New Caledonia was re- supported by a bootstrap of 72%. However, higher relationships solved as basal to the other two specimens, with support of 59.4%, between species and between genera are not resolved with any while in the Bayesian tree, the specimen from the USA was basal, degree of certainty. Support for deeper nodes were not increased making the Pacific specimens monophyletic (posterior probability by the removal of third codon positions or RY coding the data (PP) = 0.57). The ML distance between the Pacific specimens was (see Supplementary Material). 3.62% (0.71% p-distance). Carpophilus mutilatus was represented The distances between the subspecies of C. oculatus are given in by three specimens from Hawaii, Tahiti and Nauru, and had a mean Table 4. In addition, C. o. gilloglyi was shown to have a very deep split ML pairwise distance of 0.42% (0.16% mean p-distance). In both ML (16.5% mean ML pairwise distance; 7.41% mean p-distance) be- and Bayesian analyses, the Nauru specimen was basal to the other tween western specimens collected in Fiji (including Rotuma) and two specimens (bootstrap: 57.9%; PP = 0.76). The relationships of eastern populations from the Kermadec Islands to French Polynesia, these two species were not resolved in any of the analyses with including Tonga. The distinction between these two groups is any degree of support, with the exception of a C. nepos–C. davidsoni
Table 3 Rate parameters of models used in analyses.
Analysis Model likelihood f (A) f (C) f (G) f (T) C
COI nt-coded TPM3uf + I+C 8179.84071 0.27986 0.19606 0.16908 0.35499 0.68700 COI 3ex TIM2 + I+C 1660.78625 0.21536 0.22705 0.22740 0.33019 0.14900 COI 3RY TrN + C 4266.50916 0.21838 0.22453 0.23058 0.32651 0.12600 COI RY TIM3ef + C 3020.17188 0.25000 0.25000 0.25000 0.25000 0.11700 28S GTR + C 3603.59084 0.20800 0.25500 0.31500 0.22200 0.32600 ITS2 GTR 1478.1793 0.24900 0.20200 0.23200 0.31700 – f (A–C) f (A–G) f (A–T) f (C–G) f (C–T) f (G–T) Invariant COI nt-coded 0.37773 2.99728 1.00000 0.37773 2.99728 1.00000 0.428 COI 3ex 2.46898 4.07567 2.46898 1.00000 38.39180 1.00000 0.416 COI 3RY 1.00000 1.50433 1.00000 1.00000 3.06616 1.00000 – COI RY 0.00001 0.62655 1.00000 0.00001 0.33507 1.00000 – 28S 0.58770 2.09683 1.48477 0.46896 3.41398 1.00000 – ITS2 0.89063 2.24518 2.55499 0.71150 1.93285 1.00000 – 432 S.D.J. Brown et al. / Molecular Phylogenetics and Evolution 64 (2012) 428–440
Aethina concolor VAN 100 Aethina concolor VAN Conotelus sp. USA 99.5 Stelidota sp. VAN Brachypeplus sp. AUS Omosita discoidea Phenolia sp. VAN 85.6 Meligethes coracinus 69.6 Meligethes gracilis Meligethes aeneus 99.9 C. marginellus TUV C. marginellus FIJ 64.7 C. bakewelli AUS 100 C. lugubris USA C. lugubris USA C. antiquus USA C. discoideus USA Epuraea ocularis SOC 98.9 Epuraea signata NZL Epuraea signata NZL C. corticinus USA 100 C. hemipterus NZL C. hemipterus AUS Urophorus humeralis AUS C. obsoletus BIS 99.9 C. nepos NCL 59.4 C. nepos SOC C. nepos USA C. dimidiatus SOC 99.8 C. o. cheesmani VAN C. o. cheesmani VAN C. o. cheesmani VAN C. robustus BIS C. gaveni NZL 100 C. schioedtei BIS C. schioedtei BIS 98.2 C. maculatus AUS C. maculatus AUS 96.4 C. maculatus TUV 66.9 C. maculatus TUV 69.1 C. maculatus TUV 98.8 C. maculatus TUV NAU 70.6 C. maculatus C. maculatus BIS 75.363.7 C. maculatus HAW 90.6 C. maculatus SOC 62.6 C. maculatus TUV C. maculatus SAM 99.7 C. davidsoni AUS C. davidsoni AUS 100 C. mutilatus NAU 57.9 C. mutilatus HAW C. mutilatus SOC VAN 87.5 C. o. oculatus C. o. oculatus VAN C. o. oculatus VAN C. o. oculatus VAN C. o. oculatus VAN C. o. oculatus SOC C. o. oculatus TUV C. o. oculatus FIJ 66 C. o. oculatus TON C. o. oculatus TON C. o. oculatus VAN C. o. oculatus FIJ C. o. oculatus FIJ C. o. oculatus VAN C. o. oculatus VAN 71.9 C. o. oculatus VAN C. o. oculatus NAU C. o. oculatus VAN C. o. oculatus VAN C. o. oculatus VAN C. o. oculatus VAN C. o. gilloglyi TUV 97.4 FIJ 73.2 C. o. gilloglyi C. o. gilloglyi FIJ FIJ 62.5 C. o. gilloglyi C. o. gilloglyi FIJ 71.6 FIJ 81.4 C. o. gilloglyi C. o. gilloglyi FIJ C. o. gilloglyi FIJ 65.8 C. o. gilloglyi FIJ C. o. gilloglyi FIJ 0.1 C. o. gilloglyi FIJ C. o. gilloglyi FIJ 76.7 C. o. gilloglyi KER C. o. gilloglyi KER C. o. gilloglyi KER C. o. gilloglyi TUB 98 C. o. gilloglyi KER C. o. gilloglyi TUB C. o. gilloglyi SOC C. o. gilloglyi SOC C. o. gilloglyi SOC C. o. gilloglyi KER C. o. gilloglyi TON C. o. gilloglyi KER C. o. gilloglyi TUB C. o. gilloglyi SOC C. o. gilloglyi TUB 66.3 C. o. gilloglyi TUB C. o. gilloglyi TUB C. o. gilloglyi TUB
Fig. 2. ML tree of nucleotide-coded COI sequences. Bootstrap values are shown above nodes with greater than 50% support. Three letter codes signify general locality of specimens: AUS–Australia, BIS–Bismarck Islands, FIJ–Fiji, HAW–Hawaii, KER–Kermadec Islands, NAU–Nauru, NCL–New Caledonia, NZL–New Zealand, SAM–Samoa, SOC– Society Islands (French Polynesia), TON–Tonga, TUB–Austral Islands (French Polynesia), TUV–Rotuma Island (close to Tuvalu), USA–United States of America, VAN–Vanuatu. S.D.J. Brown et al. / Molecular Phylogenetics and Evolution 64 (2012) 428–440 433
Table 4 value of 78.6%. In contrast to the 28S data there was sufficient var- COI mean pairwise distances between C. oculatus subspecies. Upper triangle: iation in the marker to show intra-specific genetic structure. With- p-distances; Lower triangle: ML distances; Diagonal: ML distance/p-distance. in C. o. oculatus there was found to be a lot of structuring, though C. o. oculatus C. o. gilloglyi C. o. cheesmani no geographic influence is apparent with these analyses. These C. o. oculatus 0.0091/0.0037 0.0669 0.0921 specimens were resolved as being paraphyletic with respect to a C. o. gilloglyi 0.2063 0.0910/0.0415 0.1137 monophyletic and very homogeneous C. o. gilloglyi that shows no C. o. cheesmani 0.3959 0.4662 0/0 geographic structuring. Unexpectedly, C. schioedtei came out within C. o. oculatus. It sits on a reasonably long branch within the group and may be a case of sister group relationship being resolved in the ITS2 analysis with a homoplasy, possibly combined with insufficient taxon sampling. bootstrap value of 66%. Unfortunately, C. mutilatus is not repre- sented in this dataset. 3.4. SH tests
3.3.1.2. Bayesian analyses. Consistent with the ML analyses, C. ocul- SH-tests of topology were conducted on the COI dataset to atus was not resolved as being monophyletic on the Bayesian MCC determine the individual and combined significance of Carpophilus tree (Fig. 6). The combination of C. o. oculatus + C. o. gilloglyi formed and C. oculatus non-monophyly. ITS2 was tested for C. oculatus a strongly supported clade (PP = 0.99), whereas C. o. cheesmani was monophyly only. Results show that constraining monophyly re- found to have a weakly supported relationship with C. dimidiatus sulted in a significantly worse likelihood score for both markers (PP = 0.56). Within C. o. gilloglyi, both western and eastern clades (Table 5). The change in likelihood score of enforcing C. oculatus were well supported (both with PP = 1), however, the node linking monophyly was little different from that of forcing Carpophilus the two clades did not have strong support (PP = 0.47). The sister monophyly, despite being the result of a single change in the tree, taxa of C. o. oculatus + C. o. gilloglyi were not resolved with any de- namely the change in position of C. o. cheesmani. Likewise, the gree of support. combined changes to the tree were substantially different from Unlike ML analyses, the members of the Myothorax subgenus the optimal, but little different from the individual changes. were resolved as monophyletic (PP = 0.9), however the other sub- genera were not monophyletic, and Epuraea and Urophorus are 3.5. GMYC analysis of COI nested within Carpophilus. The latter has strong support for a rela- tionship with C. obsoletus (PP = 0.93), while the former has moder- Running GMYC with a single threshold on the Bayesian MCC tree resulted in a significantly better fit than the null model ate support for being in a clade that includes C. marginellus, C. 2 lugubris, C. bakewelli, C. discoideus and C. antiquus (PP = 0.63). (logL = 604.55, 2DL = 22.73; v test, d.f. = 4, P < 0). Using multiple thresholds did not result in a significantly better model (logL = 606.35, 2DL = 3.6; v2 test, d.f. = 12, P = 0.99), and the fol- 3.3.2. 28S rDNA lowing results use the single threshold as the standard. The model Maximum likelihood inferred a single tree with a ln likelihood with the highest likelihood inferred 34 entities, with a confidence of 3603.59084 from a GTR + C model with nucleotide frequencies interval of 14–45 entities. The entities estimated by the highest and rate parameters as shown in Table 3. likelihood model were broadly congruent with the species bound- The 28S ML analysis showed resolution at the subgeneric level, aries accepted here, though it overestimated the number of entities but did not adequately resolve lower relationships (Fig. 3). The within the C. maculatus clade (Fig. 6; leftmost, green bar). Within C. monophyly of the C. oculatus group was supported by a bootstrap oculatus, the best GMYC model recognised the eastern and western of 85%; C. o. cheesmani was sister to the other two subspecies, clades of C. o. gilloglyi as separate entities. When multiple thresh- the separation supported by a bootstrap of 84.3%, while C. o. gillo- olds were considered, GMYC greatly overestimated the number glyi was paraphyletic with respect to C. o. oculatus and did not of distinct entities, resulting in dividing C. maculatus and the east- show the geographic structure found in the mitochondrial data. Be- ern clade of C. o. gilloglyi into six entities each, and C. o. oculatus yond this, resolution within the genus was extremely poor. C. into three. gaveni, C. maculatus, C. schioedtei and C. robustus were identical in An alternative method to reporting GMYC uncertainty is the their 28S sequences and were barely different from C. davidsoni, model-weighting approach of Powell (2012). Using the results C. mutilatus and C. nepos. Carpophilus dimidiatus was shown to have from both single and multiple-threshold GMYC runs, the model- an extremely divergent 28S sequence, and a relationship with Epu- weighted average number of entities is 32.5 with a variance of raea signata was supported with a bootstrap of 73%. 63.11, from a total of 155 models. None of the models gave the ex- 28S results are much more congruent with subgeneric classifi- act taxonomic arrangment proposed here, but models that sup- cation than COI, though subgenera were not strictly monophyletic ported single C. maculatus and C. o. oculatus + C. o. gilloglyi in all cases. Species within Carpophilus s. str. came out as monophy- entities had a sum of Akaike weights (W+(comb)) of 0.056 (n = 4); letic, though it also included U. humeralis. The single species within compared with W+(subsp) = 0.087 (n = 7) where a C. o. oculatus/C. Semocarpolus, C. marginellus, was sister to, but strongly differenti- o. gilloglyi split was supported; and W+(split) = 0.26 (n = 10) where ated from the Urophorus–Carpophilus s. str. clade. Ecnomorphus C. o. gilloglyi was further subdivided into the eastern and western was paraphyletic with respect to a monophyletic Myothorax clades and C. maculatus is split into an Australian and a Pacific (excluding C. dimidiatus). clade. Results from the 2000 randomly selected trees were very vari- 3.3.3. ITS2 able. The number of clusters estimated using a single threshold Analysis of 53 ITS2 sequences produced a tree with a ML of ranged from 2 to 103. The number most commonly inferred was ln = 1478.1793 using a GTR model (Table 3). Adding a gamma 34, with 141 trees having this number of clusters. The range of distribution to the model did not result in a different topology. the number of clusters inferred using multiple thresholds was ITS2 data was only gathered for select taxa within the Myotho- smaller than the single threshold, from 2 to 84, but the inter-quar- rax subgenus. At this level it showed good variation between spe- tile range was greater (21 vs 19.25) (Fig. 5). The number most com- cies (Fig. 4). Carpophilus o. cheesmani was again shown to be a sister monly inferred was 48, with 70 trees having this number of taxon to the other C. oculatus subspecies, supported by a bootstrap clusters. The number of entities covered by the MCC confidence 434 S.D.J. Brown et al. / Molecular Phylogenetics and Evolution 64 (2012) 428–440
Phenolia sp. VAN
Conotelus sp. USA 100 Aethina concolor VAN
Stelidota sp. VAN
C. marginellus FIJ 100 C. marginellus TUV
55 55.8 C. hemipterus AUS
Urophorus humeralis FIJ 59.2 Carpophilus sp. AY310664 99 100 C. lugubris USA
Epuraea signata NZL 73.6 C. dimidiatus SOC
C. discoideus USA
C. antiquus USA 88.3 C. corticinus USA
C. nepos VAN 87.6 C. nepos NCL 59 C. mutilatus NAU 63.8 C. davidsoni AUS
C. mutilatus SOC
C. o. cheesmani VAN
TUV 72.8 C. o. oculatus 84.956.5 C. o. oculatus FIJ
0.01 84.3 C. o. oculatus TON
C. o. gilloglyi FIJ
C. o. gilloglyi FIJ
C. o. gilloglyi FIJ
C. o. gilloglyi FIJ 89.9 C. o. gilloglyi KER
C. gaveni NZL
C. maculatus TUV 60.5 C. robustus BIS
C. maculatus NCL
C. maculatus NCL
C. schioedtei BIS
C. maculatus AUS
C. maculatus AUS
C. maculatus TUV
C. maculatus TUV
C. maculatus TUV
C. maculatus TUV
C. maculatus NCL
C. maculatus AUS
Fig. 3. ML tree of 28S sequences. Bootstrap values are shown above nodes with greater than 50% support. Geographic codes as for Fig. 2. interval accounted for a large proportion of the variation in the 4. Discussion sampled trees. The proportion of the single threshold results with- in the interval was 0.697, while 0.5615 of the variation in the Data presented here demonstrate that the subspecies of C. ocul- multiple threshold models was within the interval. Likelihood ratio atus are genetically well differentiated from each other, with COI tests between single and multiple threshold models showed that model-corrected distances ranging between 20–46%. In all analy- multiple thresholds did not result in significantly better models ses, C. o. oculatus and C. o. gilloglyi emerged as sister taxa. Beyond in all but two cases. that however, relationships are unclear. Carpophilus o. cheesmani S.D.J. Brown et al. / Molecular Phylogenetics and Evolution 64 (2012) 428–440 435
C. maculatus TUV 100 C. maculatus SAM C. maculatus NAU C. maculatus AUS C. maculatus SOC 66 C. nepos NCL C. davidsoni AUS C. gaveni NZL C. o. cheesmani VAN 79.1 VAN 76.2 C. o. cheesmani C. o. cheesmani VAN 78.6 C. o. cheesmani VAN C. o. oculatus VAN 63.8 C. o. oculatus VAN C. o. oculatus VAN C. o. oculatus VAN C. o. oculatus TUV 56.6 C. o. gilloglyi KER 99.1 C. o. gilloglyi TUB C. o. gilloglyi KER C. o. gilloglyi KER C. o. gilloglyi TUB C. o. gilloglyi TUB C. o. gilloglyi TUB C. o. gilloglyi SOC C. o. gilloglyi FIJ C. o. gilloglyi TUV C. o. gilloglyi FIJ C. o. gilloglyi FIJ C. o. gilloglyi TUB 63.3 C. o. gilloglyi FIJ C. o. gilloglyi FIJ C. o. gilloglyi FIJ 0.01 C. o. gilloglyi FIJ C. o. oculatus TON C. o. oculatus FIJ C. o. oculatus VAN C. o. oculatus VAN C. o. oculatus FIJ C. o. oculatus SOC C. o. oculatus FIJ BIS 99.1 C. schioedtei 86.8 C. schioedtei BIS C. schioedtei BIS 73.2 C. o. oculatus TON C. o. oculatus TON C. o. oculatus VAN C. o. oculatus VAN C. o. oculatus FIJ C. o. oculatus VAN C. o. oculatus NAU C. o. oculatus VAN C. o. oculatus VAN
Fig. 4. ML tree of ITS2 sequences. Bootstrap values are shown above nodes with greater than 50% support. Geographic codes as for Fig. 2.
was closely related to the other two subspecies according to the nuclear datasets, but was placed well outside the oculatus–gilloglyi Table 5 clade in COI analyses. C. o. oculatus and C. o. gilloglyi are reciprocally SH test results for COI and ITS2 datasets. monophyletic in the NT-coded COI dataset, however this mono- Trees log likelihood (ln L) Difference (ln L) p-Value phyly breaks down with the modification of the COI dataset, and COI both nuclear genes show paraphyly of these two taxa. Most likely 7788 0 0.5483 Removal of third-codon positions and RY coding are usually C. oculatus monophyly 9118 1330 <0.0001 undertaken to resolve deeper phylogenetic nodes as they reduce Carpophilus monophyly 9125 1337 <0.0001 noise from faster-evolving sites and eliminate transition bias (Phil- Both 9120 1332 <0.0001 lips et al., 2004). In this instance they were not successful, with no ITS2 deeper nodes being resolved with any greater certainty than in the Most likely 1491 0 0.4870 full NT-coded dataset. However, modified datasets did resolve C. o. C. oculatus monophyly 1529 38 0.0046 gilloglyi as being paraphyletic with respect to C. o. oculatus,an observation that is consistent with 28S data. This paraphyly hints 436 S.D.J. Brown et al. / Molecular Phylogenetics and Evolution 64 (2012) 428–440 Frequency Frequency 0204060 04080140 0 20406080100 0 20406080 Number of clusters Number of clusters
Fig. 5. Frequency of the number of clusters estimated by GMYC models for 2000 randomly selected trees from the Bayesian MCMC chain. Solid vertical lines show the confidence interval of the number of clusters estimated by GMYC on the MCC tree. Left: single GMYC threshold; Right: Multiple GMYC threshold. that incomplete linage sorting may be affecting the gene trees pre- always been considered to be distinct, usually being placed in a dif- sented here, or suggests an ancestor–descendant relationship be- ferent subfamily to Carpophilus. Urophorus was elevated to a full tween C. o. gilloglyi and C. o. oculatus. Removal of third-codon genus by Gillogly (1962), but later workers have debated the valid- positions also led to a decrease in genetic structuring within C. o. ity of this elevation and have continued to consider it as a subge- gilloglyi and C. o. oculatus, showing that the bulk of the differences nus (e.g. Ewing and Cline, 2005). Though the structure of the between these two taxa are in the faster-evolving third-codon po- male terminalia in U. humeralis looks similar to that in Carpophilus, sition, while differences between these taxa and C. o. cheesmani in- closer investigation shows that the 8th tergite makes up the entire clude first and second codon positions, confirming that C. o. pygidium in U. humeralis, as opposed to the small, ventrally situ- cheesmani is an older lineage than the other two taxa. ated button-like struction found in Carpophilus. Urophorus pos- Despite having genitalia and colouration very similar to C. o. sesses a round patch with significantly different sculpture in the gilloglyi, C. maculatus was not inferred as being the sister taxon to same location as the 8th tergite in Carpophilus, which completes C. oculatus. COI analyses did not show any support for nodes the illusion of similarity. This morphological feature provides sig- beyond species-level groupings, while analysis of 28S resolved a nificant evidence against the placement of Urophorus within Carpo- sister group relationship with a clade including C. maculatus, philus as inferred from our molecular data. Greater taxon sampling C. robustus, C. schioedtei and C. gaveni. within Urophorus and other Carpophilus subgenera and the use of a While the sample sizes and geographic coverage of other Carpo- genetic marker with a conservative rate of evolution is needed to philus species are not large enough to make definitive conclusions investigate this situation further. regarding their evolutionary history, it is interesting to note the Despite being proposed as a nuclear equivalent of the COI re- comparative amounts of variation in C. nepos, C. mutilatus and C. gion for the identification of species (Sonnenberg et al., 2007), maculatus. Carpophilus nepos shows distinct differences in COI be- the 28S D1–D2 region proved to be unreliable for distinguishing tween the three specimens from Tahiti, New Caledonia and from between species-level taxa in this research. This was particularly a laboratory culture in the USA, with average intra-specific ML dis- the case within the Myothorax subgenus, members of which were tances of 4.2%. This degree of genetic variation suggests that this distinctly different in their COI sequences. However, 28S did show species may have been present in the Pacific for some time. By promise for the detection of higher-level phylogenetic relation- comparison, C. mutilatus specimens showed a much lower level ships. The placement of U. humeralis and Epuraea within Carpophi- of variation with an average ML intra-specific distance of 0.42%, de- lus may be explained by insufficient taxon sampling or long branch spite specimens being collected over a similarly large geographic attraction. The level of divergence of C. dimidiatus is very high. area. This much lower level of genetic variation suggests that C. More specimens of this species are required to confirm whether mutilatus may be a relatively recent arrival in the Pacific compared this divergence is an error. As a member of the Myothorax subge- with C. nepos, or that it may be more readily dispersed, resulting in nus, it is expected that the species would have been placed with greater genetic connectivity between populations. Carpophilus the remainder of species in that subgenus. maculatus shows genetic structure between island groups and GMYC results were largely congruent with morphological spe- within single islands, suggesting that this species has been present cies. In some instances the model with the highest likelihood over- in these areas for a substantial period of time. The greatest degree estimated the number of species when intra-specific genetic of structuring exists between a clade representing two Australian structuring was evident, i.e. within C. o. oculatus + C. o. gilloglyi specimens and a clade composed of Pacific Islands specimens; and C. maculatus. However, models within the confidence interval these two groups differing by a genetic distance of over 10%. This of the analysis were more inclusive. The GMYC confidence interval separation is not shown by the nuclear genes sampled, and no of the MCC tree adequately reflected the variability in the sample, other evidence currently exists that would justify considering as reflected by the confidence interval encompassing the bulk of these populations different species. The level of genetic structuring the density of the distribution of the results on the sampled trees. may represent long isolation of the Australian and Pacific popula- The use of the AIC and model weights extends the use of GMYC from tions of C. maculatus. its current use as comparing two models, to enabling the weight of Species assigned to the subgenus Myothorax formed a mono- evidence for different scenarios to be considered in taxonomic deci- phyletic group in most analyses, though in COI data it was present sions. In this instance, while there is more evidence from GMYC for with low support. 28S was reasonably congruent with subgeneric recognising the East/West split in C. o. gilloglyi and the Australia/ classifications, but did not resolve subgenera as being strictly Pacific split in C. maculatus, the evidence ratios against recognising monophyletic. These preliminary results suggest that the classifi- the subspecies (Esplit,subsp = 2.981), or having a combined C. o. ocula- cation of Kirejtschuk (2008) may represent natural clades, however tus + C. o. gilloglyi clade (Esplit,comb = 4.604) is not so high as to be be- more comprehensive species sampling within these subgenera is yond the realms of possibility. GMYC was originally developed to required before this can be confirmed. provide estimates of species numbers as part of large-scale commu- Our molecular data consistently resolve Urophorus and Epuraea nity ecology projects where in-depth taxonomic expertise was not within Carpophilus sensu lato. While the first of these genera was available and not necessary. In this context it can be a useful tool originally described as a subgenus of Carpophilus, the second has (Monaghan et al., 2009; Pinzon-Navarro et al., 2010); however its S.D.J. Brown et al. / Molecular Phylogenetics and Evolution 64 (2012) 428–440 437
1 C. marginellus FIJ 0.46 C. marginellus TUV 0.88 C. bakewelli AUS 1 USA 0.63 C. lugubris C. lugubris USA 0.33 C. discoideus USA 0.26 C. antiquus USA 0.91 Epuraea ocularis SOC 1 Epuraea signata NZL 0.25 Epuraea signata NZL 0.03 Stelidota sp. VAN 0.82 Brachypeplus sp. AUS 0.41 Conotelus sp. USA 1 VAN 0.56 Aethina concolor Aethina concolor VAN 0.66 Omosita discoidea 0.47 Phenolia sp. VAN 1 1 Meligethes coracinus 1 Meligethes gracilis Meligethes aeneus 0.93 C. obsoletus BIS 0.56 Urophorus humeralis AUS 0.83 C. corticinus USA 1 C. hemipterus NZL C. hemipterus AUS 0.56 C. dimidiatus SOC 0.76 1 C. o. cheesmani VAN 0.33 C. o. cheesmani VAN C. o. cheesmani VAN 1 C. nepos USA 0.57 C. nepos NCL 0.9 C. nepos SOC 1 C. mutilatus NAU 0.76 C. mutilatus HAW C. mutilatus SOC 1 C. davidsoni AUS 0.14 C. davidsoni AUS 1 C. schioedtei BIS 0.11 0.64 C. schioedtei BIS 0.35 C. gaveni NZL BIS 0.71 C. robustus 1 C. maculatus AUS C. maculatus AUS 0.49 1 0.95 C. maculatus TUV 0.22 C. maculatus TUV 10.99 C. maculatus TUV C. maculatus TUV 0.73 C. maculatus NAU 0.94 C. maculatus BIS 1 C. maculatus SAM 0.42 C. maculatus TUV 0.99 C. maculatus HAW C. maculatus SOC 0.99 C. o. oculatus TON 0.11 0.21 C. o. oculatus TON 0.16 C. o. oculatus TUV 0.460.15 C. o. oculatus FIJ C. o. oculatus SOC C. o. oculatus VAN 0.220.7 C. o. oculatus VAN 0.961 C. o. oculatus VAN 0.22 C. o. oculatus VAN C. o. oculatus VAN C. o. oculatus FIJ 0.46 C. o. oculatus FIJ 0.170.08 C. o. oculatus VAN 0.12 C. o. oculatus VAN C. o. oculatus VAN 0.080 C. o. oculatus VAN 0 C. o. oculatus VAN 0.08 C. o. oculatus VAN 0.99 0.01 C. o. oculatus NAU 0.08 C. o. oculatus VAN C. o. oculatus VAN C. o. gilloglyi TUV 1 FIJ 0.8 C. o. gilloglyi C. o. gilloglyi FIJ 0.1 C. o. gilloglyi FIJ 0.64 C. o. gilloglyi FIJ 0.960.03 C. o. gilloglyi FIJ 0.11 C. o. gilloglyi FIJ 0.78 C. o. gilloglyi FIJ 0.47 0.02 C. o. gilloglyi FIJ 0.03 C. o. gilloglyi FIJ 0.1 0.11 C. o. gilloglyi FIJ C. o. gilloglyi FIJ 1 C. o. gilloglyi KER 0.33 C. o. gilloglyi KER C. o. gilloglyi KER C. o. gilloglyi SOC 0.2510.96 C. o. gilloglyi TUB 0.23 C. o. gilloglyi KER 0.36 C. o. gilloglyi SOC 0.140.53 C. o. gilloglyi SOC C. o. gilloglyi SOC 0.2 C. o. gilloglyi KER TON 0.03 C. o. gilloglyi 0.07 C. o. gilloglyi KER 0 C. o. gilloglyi TUB 0.15 C. o. gilloglyi TUB 0.83 C. o. gilloglyi TUB 1 C. o. gilloglyi TUB 0.34 C. o. gilloglyi TUB C. o. gilloglyi TUB
Fig. 6. Bayesian maximum clade credibility tree of NT-coded COI sequences. Posterior probabilities are shown above all nodes. 95% highest posterior density error bars for node heights are given for nodes with a posterior probability greater than 0.5. Geographic codes are the same as in Fig. 2. Bars to the right of the tree show the composition of clusters estimated by single-threshold GMYC models (from left to right): highest likelihood GMYC model (green), the lower confidence interval (light blue), and the upper confidence interval (blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 438 S.D.J. Brown et al. / Molecular Phylogenetics and Evolution 64 (2012) 428–440 estimates are very broad, thereby limiting its utility in the context rate of evolution, as COI showed saturation at the subgenus level of a systematic treatment of a taxon. within Carpophilus. A somewhat more robust result is that the sub- In the light of the morphological and genetic differences be- genus Myothorax appears to be monophyletic. Unfortunately, no tween the subspecies of C. oculatus, it is justifiable to elevate C. o. other Carpophilus subgenera showed any indication of monophyly, cheesmani to a full species while retaining C. o. gilloglyi and C. o. but the sampling scheme was not designed to explicitly test this oculatus as subpecies. In all analyses, C. o. cheesmani is monophy- hypothesis. letic and substantially different from other C. oculatus subspecies, Finally, this study further confirms that the South Pacific region and can separated by the shape of male genitalia, and by the differ- is a dynamic region, and shows that widespread species within the ences in pronotal punctation (Dobson, 1993b). The elevation of this region can have intruiging biology and can be as interesting and subspecies to full species follows the precedent of a Californian ti- informative as single island endemics. ger beetle, Cicindela lunalonga Schaupp, which was elevated from being a subspecies of Ci. terricola following molecular evidence of 5.1. Taxonomy of C. maculatus and C. oculatus sensu lato strict monophyly and a high pairwise divergence in phylogenetic analyses (Woodcock et al., 2007). The two other subspecies of C. The length of specimens were measured from the anterior mar- oculatus are retained at that rank as they show paraphyly, and as gin of the pronotum to the posterior margin of the elytra at the ely- an indication that this complex has some interesting biology wor- tral suture, as the abdomen in preserved specimens can be thy of further study. significantly distended or contracted. Specimens were received There may be some justification for the species determination of on loan from the Oxford University Museum of Natural History the C. o. gilloglyi geographic clades. However, no lines of evidence (Oxford, Britain) (OUMNH), New Zealand Arthropod Collection outside of COI currently point to species-level differences between (Auckland, New Zealand) (NZAC), MAF Biosecurity New Zealand the two clades. Specimens from these areas, which appear to be (Auckland, New Zealand) (MAF), Queensland Museum (Brisbane, identical morphologically, have been found in the same habitat Australia) (QM), the University of the South Pacific (Suva, Fiji) and appear to have the same ecological niche. It is possible that (USP) and the Hunterian Museum (Glasgow, Scotland) (HM). these populations are in the early stages of allopatric speciation. It is surprising that these two clades are so distinct considering 5.1.1. Carpophilus maculatus (Murray, 1864) the high frequency of human movement between Fiji and Tonga. Diagnosis: L: 1.6–2.2 mm, W: 1–1.4 mm. Colour variable from It is even more puzzling when the large geographical distances be- yellow–brown to dark brown, often with lighter, T-shaped marking tween islands inhabited by the eastern clade are considered, over on the elytra. Pronotum length/width ratio 1:1.5, punctured with which it appears to have maintained recent gene flow. Because of reniform punctures (Fig. 7a). Prosternal process expanded behind the limited sampling in Tonga, more focused collecting throughout procoxae. Female pygidium truncate. Tonga and the Lau archipelago would be needed to determine Distribution: Bismarck Archipelago (Gillogly, 1969, LUNZ), Car- whether the apparent geographic separation of the two C. o. gilloglyi oline Islands (Gillogly, 1962), Cook Islands (NZAC, Dobson un- clades is real, or if there is some overlap in the ranges of these clades publ., LUNZ), Easter Island (HM), Fiji (MAF, Dobson unpubl., and to what extent that overlap occurs. More detailed specimen NZAC, LUNZ), Gilbert Islands (Dobson unpubl., Gillogly, 1962), sampling, particularly with the use of rapidly evolving genetic Guam (Gillogly, 1962), Hawaii (Ewing and Cline, 2005; Murray, markers such as microsatellites, may provide clues to the creation 1864; Nishida and Beardsley, 2002), West Papua (Dobson unpubl.), and persistance of this deep divergence within C. o. gilloglyi. Should Kiribati (Tarawa) (NZAC), Marquesas Islands (Dobson unpubl.), the two clades be found to exist in sympatry, the proportion of indi- Mariana Islands (Gillogly, 1962), Marshall Islands (Gillogly, viduals belonging to each clade plus the genetic diversity of these 1962), Nauru (LUNZ), New Caledonia (NZAC, LUNZ, Dobson un- two populations and extent to which their distributions overlap publ., QM), Niue (NZAC, Dobson unpubl., HM), Palau (Gillogly, may offer clues as to the processes (e.g. invasion) that currently 1962), Papua New Guinea (Dobson unpubl.), Samoa (NZAC, LUNZ, influence the distribution of each clade. Dobson unpubl., HM), Society Islands (LUNZ, Dobson unpubl.), Sol- omon Islands (Dobson unpubl.), Tokelau (NZAC, HM), Tonga 5. Conclusion (NZAC, MAF, Dobson unpubl.), Tuamotu Archipelago (Dobson un- publ.), Austral Islands (Dobson unpubl., LUNZ), Tuvalu (NZAC, This research set out to resolve the taxonomic questions sur- LUNZ, HM), Vanuatu (Dobson unpubl., OUMNH, NZAC, LUNZ). rounding the subspecies of C. oculatus, and to determine the degree Christmas Is., Australia, Philippines, Cuba, Cocos-Keeling Is, of similarity between C. oculatus and C. maculatus. Consistent Indonesia, Nicobar Islands, Mollucas. evidence from three gene regions reveal that C. maculatus and Comments: Abundant throughout the Pacific and found on a C. o. cheesmani are distinct species, while C. o. oculatus and wide variety of fruit and vegetables. Very variable in colouration. C. o. gilloglyi are closely related taxa that show paraphyletic struc- This species be distinguished from C. oculatus and C. cheesmani by ture with genetic markers. These species therefore need to be trea- the wider pronotum. Illustration of the male genitalia can be found ted as such in biosecurity operations. While this distinction will in Dobson (1955). necessitate increased vigilance by biosecurity organisations, the level of genetic structuring between islands of the South Pacific 5.1.2. Carpophilus cheesmani Dobson 1993b stat. rev. suggests that C. oculatus and C. maculatus may not be as invasive Diagnosis: L: 1.8–2.4 mm, W: 1.0–1.4 mm. Colour dark brown as previously assumed. to black, with lighter, reddish ring on each elytron. Pronotum At a higher level, this study suggests that Carpophilus may not length/width ratio 1:1.3, coarsely and densely punctured with be monophyletic, potentially incorporating the genera Urophorus reniform punctures (Fig. 7b), microsculpture minutely reticulate and Epuraea. This is unexpected as it runs against several lines of coriarious. Prosternal process rounded. Female pygidium truncate. evidence from morphology, but is to be taken with caution as Distribution: Vanuatu (Malekula, Ounua, Tanna, Erromango, few specimens and species of Urophorus and Epuraea were sampled Efate) (Dobson, 1993b, LUNZ). for this study, and these taxa appear with the representatives of a Comments: Restricted to Vanuatu where it is sympatric with poorly sampled subgenus of Carpophilus. A formal investigation of C. o. oculatus from which it can be differentiated by the sculpture this possibility will require representative samples of all three of the pronotum. The two species can be found syntopically, genera. It will also need to use genetic markers that have a slower however C. cheesmani is the rarer of the two (SDJ Brown pers. S.D.J. Brown et al. / Molecular Phylogenetics and Evolution 64 (2012) 428–440 439
Fig. 7. Habitus photographs of Carpophilus maculatus, C. cheesmani and C. oculatus, showing detail of pronotum. (a) C. maculatus Rarotonga, Cook Islands. (b) C. cheesmani Efate, Vanuatu. (c) C. o. oculatus Tongatapu, Tonga. (d) C. o. gilloglyi Rapa, Austral Islands, French Polynesia.
obs.). This species has been collected from the rotting fruit of bread- Eua, Niuafo’ou) (Dobson, 1993b, NZAC, MAF, HM, LUNZ), Austral fruit (Artocarpus altitis), oranges (Citrus sp.) and guava (Psidium guaj- Islands (Rapa, Rimatara) (LUNZ), Tuvalu (Rotuma) (LUNZ). ava), and sweet potato (Ipomoea batatas) tubers. Comments: Widespread throughout the South Pacific and found on a wide variety of fruit and vegetables. While the ranges of C. o. 5.1.3. Carpophilus oculatus oculatus (Murray, 1864) oculatus and C. o. gilloglyi overlap over a significant part of their Diagnosis: L: 1.8–2.6 mm, W: 1.0–1.6 mm. Colour variable, from range, they are rarely found syntopically (SDJ Brown pers. obs.). light brown to black, usually with lighter ring on each elytron. Pronotum length/width ratio 1:1.3, pronotal punctures sparse, round and fine on disc, becoming reniform and coarser toward Acknowledgments margin (Fig. 7c), no impunctate medial line; microsculpture min- utely reticulate coriarious; usually less pubescent. Female pygid- Nick Porch (Deakin University, Australia), Mofakhar Hossein ium truncate. (Department of Primary Industries, Victoria, Australia), Kiteni Kuri- Distribution: Cook Islands (Rarotonga) (Dobson, 1993b, NZAC), ka (NARI, Papua New Guinea), Christian Mille (IAC, New Caledo- Fiji (Quisenberry, Taveuni, Kadavu, Viti Levu, Vanua Levu) (Dobson, nia), Linette Berukilukilu (VQIS, Vanuatu), Sada Nand Lal (SPC, 1993b, LUNZ, MAF, USP, NZAC), Hawaii (Dobson, 1993b), Marque- Fiji), Will Haines (University of Hawaii), Katharina Pötz (Denmark), sas Islands (Fatu Hiva) (Dobson, 1993b), Nauru (LUNZ), New Cal- Maja Poeschko (Ministry of Agriculture, Cook Islands), Kuateane edonia (Grande Terre) (QM, NZAC), Niue (Niue) (NZAC), Samoa Tupola (Ministry of Agriculture, Samoa) and Rudolph Putoa (Upolu, Tutuila) (HM, NZAC), Society Islands (Bora Bora, Tahiti) (Département de la Recherche Agronomique, Tahiti) provided (Dobson, 1993b, LUNZ), Tokelau (Nukunono) (NZAC), Tonga (Ton- specimens for DNA sequencing. Museum specimens were loaned gatapu) (Dobson, 1993b, NZAC, MAF, HM, LUNZ), Tuvalu (Rotuma) by James Hogan (Oxford University Museum of Natural History, (LUNZ), Vanuatu (Espiritu Santo, Efate) (LUNZ). UK), Shepherd Myers (Bishop Museum, Hawaii), Geoff Monteith Comments: Widespread throughout the South Pacific and found (Queensland Museum, Australia), Geoff Hancock (Hunterian Mu- on a wide variety of fruit and vegetables. seum, UK), Olwyn Green (MAF Biosecurity, New Zealand) and Hilda Waqa (University of the South Pacific, Fiji). Ron Dobson (Glasgow) 5.1.4. Carpophilus oculatus gilloglyi Dobson 1993b provided unpublished distribution records of South Pacific Carpo- Diagnosis: L: 1.5–2.4 mm, W: 0.9–1.4 mm. Colour variable, from philus. Alexander Kirejtschuk (Russia) assisted in identifying C. light brown to black, usually with lighter ring on each elytron. robustus and C. schioedtei. Curtis Ewing (UC Berkeley, United States Pronotum length/width ratio 1:1.3, pronotal punctures on vertex of America) allowed use of unpublished Carpophilus sequences. and disc of pronotum close and reniform (Fig. 7d), impunctate Rich Leschen (Landcare Research, New Zealand), Rupert Collins median line anterior of scutellum; microsculpture reticulate coria- (Bio-Protection Research Centre, New Zealand) and John Marris rious, coarser than in other subspecies. Female pygidium emargin- (Lincoln University, New Zealand) provided valuable comments ate medially. on the research and manuscript. Distribution: Caroline Islands (Ponape Island, Truk) (Dobson, 1993b), Cook Islands (Rarotonga, Atiu) (Dobson, 1993b, NZAC, MAF), Easter Island (NZAC), Fiji (Vanua Levu, Taveuni, Viti Levu, Kadavu, Ovalau, Lakeba) (LUNZ, Dobson, 1993b, USP, NZAC), Appendix A. Supplementary material Kermadec Islands (Meyer Island, Raoul Island, Chanters Islets) (Dobson, 1993b, NZAC, LUNZ), Niue (Dobson, 1993b, NZAC), Supplementary data associated with this article can be found, in Samoa (Upolu, Savai’i) (NZAC, HM), Society Islands (Bora Bora, the online version, at http://dx.doi.org/10.1016/j.ympev.2012. Moorea, Tahiti) (Dobson, 1993b, LUNZ), Tonga (Tongatapu, Vava’u, 04.018. 440 S.D.J. Brown et al. / Molecular Phylogenetics and Evolution 64 (2012) 428–440
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