Daniel Rubinoff

Myth of the molecule: DNA barcodes for species cannot replace morphology for identification and classification

So‐called DNA barcodes have recently been proposed to answer the problem of specimen identification and to quantify global biodiversity. We show that this proposition is wanting in terms of rationale, methodology and interpretation of results. In addition to falling short of all its stated goals, the method abandons the benefits of morphological studies in favor of a limited molecular identification system that would ultimately impede our understanding of biodiversity.

Vertical Transmission of a Pentatomid Caeca-Associated Symbiont

Abstract We present molecular data for an endosymbiont of the insect family Pentatomidae, located in the gastric caeca of Nezara viridula (L.) (Hemiptera: Pentatomidae) stink bugs. Restriction fragment length polymorphism and polymerase chain reaction analysis suggest that this bacterium is consistently present in caeca of N. viridula from a variety of geographic locations. The bacterium is present in different midgut sections in nymphs versus adults. The bacterium also was detected on eggshells after nymphs had hatched but not in ovarioles, suggesting oral rather than transovarial transmission. Surface sterilization of egg masses generated aposymbiotic insects. Aposymbiotic individuals reached the adult stage, females laid viable eggs, and the offspring remained aposymbiotic in the following generation. No clear fitness decrease was observed in aposymbiotic individuals over two generations. Phylogenetic analysis of a partial 16S rRNA data set with 21 Gammaproteobacteria suggested the inadequacy of neighbor-joining and maximum parsimony models to account for homoplasy apparent in a molecular data set, including a range of insect endosymbionts. Maximum likelihood-based analysis suggests that the N. viridula endosymbiont is closely related to a caeca-associated symbiont found in another stink bug family (Plataspidae). The high AT content of the symbiont’s 16S rRNA in relation to other insect endosymbionts, its location in the midgut of the host insect, oral transmission, and survival of aposymbiotic individuals suggest this symbiosis may be recently established.

Who Will Actually Use DNA Barcoding and What Will It Cost

It is likely that the mere use of the word barcode is responsible for much of the appeal surrounding DNA barcoding, after all DNA-based identification methods (e.g., DeSalle and Birstein, 1996) used prior to Hebert et al.’s (2002) proposal of the term failed to ignite significant attention from the scientific community and none whatsoever from the general public. The term itself is loaded. Product barcodes are scanned using checkout lasers and indeed the image of the “Star Trek tricorder,” a handheld scanner, has been used repeatedly by barcoding proponents in both presentations and papers (Janzen, 2004; Smith, 2005). Savolainen et al. (2005) use just such an allusion to commence their introductory paper of a special issue of Philosophical Transactions of the Royal Society of London devoted to DNA barcoding. Although the need for scanners capable of detecting biological weapons will undoubtedly lead to the development of portable DNA scanners at some point (a stated objective of the U.S. Department of Homeland Security, Directorate for Science and Technology), there is, however, absolutely no indication that they will be affordable or practical for the kinds of mass identification uses that barcoding proponents are selling to the general public as the outcome of this research. Further, a critical difference between a “tricorder” and a portable DNA barcoder would be the DNA component. Whereas Mr. Spock need only wave his tricorder in the general direction of an alien to be told what it is, real-world barcoders will need to actually handle that animal, remove tissue from it, and load it into the barcoder to get an identification. DNA barcoding is intrinsically linked to specimens as samples that must be collected for DNA extraction, be it in a molecular biology laboratory as at present or by a handheld barcoder at some point in the future. This small detail neatly circumscribes what barcoding can, and what it can’t, achieve. Who then would use DNA barcoding? Dan Janzen has written eloquently and spoken passionately about the need to improve biological literacy amongst the general public and that without the ability to “read” nature by identifying its contents, biodiversity is doomed to be underappreciated and so destroyed (see Janzen et al., 2005, for full development of this argument). One wonders what the place for even a portable DNA barcoder is in this vision. The majority of the public observes nature; they don’t sample it by removing the legs from butterflies and throwing them into a barcoder to get identifications. Wildlife protection authorities expend a lot of effort trying to keep people away from wildlife and certainly not handling or vivisecting them. Will the advent of a portable barcoder result in a complete turn around by wildlife authorities encouraging the wholesale handling of wildlife by the public? For smaller or more delicate plants and animals (which would include those most difficult for an interested amateur to identify without a barcode), being “identified” with a barcoder is likely to be fatal. Not simply due to the handling or picking necessarily, but due to the dissection necessary to get a tissue sample. Further, such barcode identifications will not share the benefits of traditionally collected specimens because an inexperienced member of the public is likely to be simply, and passingly curious, and not inclined to retain each “specimen” as a voucher with rigorous locality data for future study or reference. Although proponents suggest to compensate new species vouchers with free identifications (Janzen, 2004), one needs only think of the vast majority of people who toss aside cans they could recycle for a refund. The inconvenience of handling and sending a specimen in decent condition is far greater than recycling. Clearly such barcodes are unlikely to inspire or benefit the vast majority that needs to be reached to protect biodiversity. Amongst the wider public, the largest group identifying species on a regular basis is birdwatchers. DNA barcoding will be of limited use to them unless it is proposed that a 12-gauge shotgun now become standard equipment for such “twitchers.” Even for professional bird identifications, such as those prepared for biological impact studies, trapping is secondary to visual identifications due to the stress that handling inflicts on birds. Mammal identification for the general public is based on direct visual identifications as well as the interpretation of trail signs, scats, or hair. Perhaps some identifications could come through

Phylogeography and ecology of an endemic radiation of Hawaiian aquatic case-bearing moths (Hyposmocoma: Cosmopterigidae)

The endemic moth genus Hyposmocoma (Lepidoptera: Cosmopterigidae) may be one of the most speciose and ecologically diverse genera in Hawaii. Among this diversity is the Hyposmocoma saccophora clade with previously unrecorded aquatic larvae. I present a molecular phylogeny based on 773 base pairs (bp) of the mitochondrial gene cytochrome c oxidase subunit I and 762 bp of the nuclear gene elongation factor 1-α. Topologies were constructed from data using maximum-parsimony, maximum-likelihood and Bayesian search criteria. Results strongly support the monophyly of the H. saccophora clade and the monophyly of the genus Hyposmocoma. The H. saccophora clade has single-island endemic species on Oahu, Molokai and West Maui. By contrast, there are three species endemic to Kauai, two being sympatric. The H. saccophora clade appears to follow the progression rule, with more basal species on older islands, including the most basal species on 11 Myr-old Necker Island, one of the Northwestern Hawaiian Islands. Aquatic behaviour either evolved recently in the species on the main Hawaiian Islands or was secondarily lost on the arid northwestern Necker Island. The phylogeny suggests that Hyposmocoma is older than any of the current main islands, which may, in part, explain Hyposmocomas remarkable diversity.

An Evaluation of the Species Status of Bactrocera invadens and the Systematics of the Bactrocera dorsalis (Diptera: Tephritidae) Complex

ABSTRACT The genus Bactrocera (Tephritidae) contains >500 species, including many severe pests of fruits and vegetables. Although native to tropical and subtropical areas of Africa, India, Southeast Asia, and Australasia, a number of the pest species, largely members of the Bactrocera dorsalis (Hendel) complex, have become widespread through accidental introduction associated with agricultural trade. The B. dorsalis complex includes several morphologically and ecologically similar pests, making species designations uncertain. One of these, Bactrocera invadens Drew, Tsuruta, and White, endemic to Sri Lanka, has spread across Africa in the last decade and become a major agricultural pest. We sequenced one mitochondrial and two nuclear genes from 73 specimens, belonging to 19 species to construct phylogenies and examine species relationships and limits within the genus Bactrocera and several species of the B. dorsalis complex-specifically addressing the placement of B. invadens. Results indicate the B. dorsalis complex is polyphyletic. B. invadens and several other species within the B. dorsalis complex (B. dorsalis, Bactrocera papayae Drew &Hancock, and Bactrocera philippinensis Drew & Hancock) are also paraphyletic with respect to each other and probably represent a single genetically indistinguishable, phenotypically plastic, pest species that has spread throughout the world.

Multiple aquatic invasions by an endemic, terrestrial Hawaiian moth radiation

Insects are the most diverse form of life on the planet, dominating both terrestrial and freshwater ecosystems, yet no species has a life stage able to breath, feed, and develop either continually submerged or without access to water. Such truly amphibious insects are unrecorded. In mountain streams across the Hawaiian Islands, some caterpillars in the endemic moth genus Hyposmocoma are truly amphibious. These larvae can breathe and feed indefinitely both above and below the waters surface and can mature completely submerged or dry. Remarkably, a molecular phylogeny based on 2,243 bp from both nuclear (elongation factor 1α and carbomoylphosphate synthase) and mitochondrial (cytochrome oxidase I) genes representing 216 individuals and 89 species of Hyposmocoma reveals that this amphibious lifestyle is an example of parallel evolution and has arisen from strictly terrestrial clades at least three separate times in the genus starting more than 6 million years ago, before the current high islands existed. No other terrestrial genus of animals has sponsored so many independent aquatic invasions, and no other insects are able to remain active indefinitely above and below water. Why and how Hyposmocoma, an overwhelmingly terrestrial group, repeatedly evolved unprecedented aquatic species is unclear, although there are many other evolutionary anomalies across the Hawaiian archipelago. The uniqueness of the community assemblages of Hawaiis isolated biota is likely critical in generating such evolutionary novelty because this amphibious ecology is unknown anywhere else.

Ancient diversification of Hyposmocoma moths in Hawaii

Island biogeography is fundamental to understanding colonization, speciation and extinction. Remote volcanic archipelagoes represent ideal natural laboratories to study biogeography because they offer a discrete temporal and spatial context for colonization and speciation. The moth genus Hyposmocoma is one of very few lineages that diversified across the entire Hawaiian Archipelago, giving rise to over 400 species, including many restricted to the remote northwestern atolls and pinnacles, remnants of extinct volcanoes. Here, we report that Hyposmocoma is ~15 million years old, in contrast with previous studies of the Hawaiian biota, which have suggested that most lineages colonized the archipelago after the emergence of the current high islands (~5 Myr ago). We show that Hyposmocoma has dispersed from the remote Northwestern Hawaiian Islands to the current high islands more than 20 times. The ecological requirements of extant groups of Hyposmocoma provide insights into vanished ecosystems on islands that have long since eroded.

Evolution of ecological traits and wing morphology in Hemileuca (Saturniidae) based on a two-gene phylogeny

We present a molecular phylogeny for the genus Hemileuca (Saturniidae), based on 624 bp of mitochondrial cytochrome oxidase I (COI) and 932 bp of the nuclear gene elongation factor 1 alpha (EF1alpha). Combined analysis of both gene sequences increased resolution and supported most of the phylogenetic relationships suggested by separate analysis of each gene. However, a maximum parsimony (MP) model for just COI sequence from one sample of most taxa produced a phylogeny incongruent with EF1alpha and combined dataset analyses under either MP or ML models. Time of year and time of day during which adult moths fly corresponded strongly with the phylogeny. Although most Hemileuca are diurnal, ancestral Hemileuca probably were nocturnal, fall-flying insects. The two-gene molecular phylogeny suggests that wing morphology is frequently homoplastic. There was no correlation between the primary larval hostplants and phylogenetic placement of taxa. No phylogenetic pattern of specialization was evident for single hostplant families across the genus. Our results suggest that phenological behavioral characters may be more conserved than the wing morphology characters that are more commonly used to infer phylogenetic relationships in Lepidoptera. Inclusion of a molecular component in the re-evaluation of systematic data is likely to alter prior assumptions of phylogenetic relationships in groups where such potentially homoplastic characters have been used.

A revised molecular phylogeny of the globally distributed hawkmoth genus Hyles (Lepidoptera: Sphingidae), based on mitochondrial and nuclear DNA sequences.

The hawkmoth genus Hyles comprises some 29 species with a global distribution. In this study, we augment the previous taxon sampling with more species and add sequences from a nuclear gene to produce a refined phylogenetic hypothesis. A total evidence reconstruction based on Bayesian analysis of the combined mitochondrial (COI, t-RNA-Leu, COII; 2284 bp) and nuclear (EF1alpha; 773 bp) sequences is discussed and compared with the results from separate analyses of the two genes. The total evidence phylogeny corroborates many of the phylogenetic relationships previously postulated within the genus. In addition, the hitherto unsampled enigmatic species Hyles biguttata from Madagascar appears as sister group to Hyles livornicoides from Australia, although support for the relationship is relatively weak. The high level of differentiation of Hyles perkinsi from H. calida (both Hawaii), and the status of these two as sister species, is corroborated by both sources of sequence data. However, their phylogenetic position when mt DNA sequences alone are considered differs markedly from that under total evidence. The previously postulated relationships within the Hyles euphorbiae complex (HEC) s.s. are largely corroborated, but H. dahlii is now more closely related and the HEC s.l. is redefined to include H. zygophylli and H. stroehlei (two species that had not been studied previously using molecular data) and to exclude H. siehei and H. hippophaes. The nuclear sequences alone are insufficiently variable to fully resolve all lineages and the phylogeny suggests that nuclear gene swapping and incomplete lineage sorting have occurred implying recent divergence. The results from the total evidence analysis provide a phylogenetic hypothesis that both corroborates and complements the previous biogeographic scenario, and provides new insights into the origins of several of the included taxa.

Vagility across Vanessa (Lepidoptera: Nymphalidae): mobility in butterfly species does not inhibit the formation and persistence of isolated sister taxa

Allopatric speciation is thought to occur in the absence of gene flow, thereby suggesting that widespread vagile species might be less likely to generate restricted sister taxa because of a lack of isolation. The butterfly genus Vanessa provides an ideal test of this concept, as it contains some of the most cosmopolitan and vagile species of butterflies on the planet, as well as some highly restricted taxa. Given the age of these groups, this arrangement offers a special opportunity to examine the relationship between vagility and phylogeny in generating novel taxa; specifically, does the vagility of some lineages impede allopatric speciation, leaving restricted clades more speciose? A phylogenetic hypothesis is proposed for all species belonging to the butterfly genus Vanessa based on DNA sequences from one mitochondrial and eight nuclear gene regions. The resulting topology shows very little conflict among gene regions, with five well‐supported clades corresponding to morphologically consistent species groups. The data very strongly indicate a polyphyletic genus Antanartia, and thus to preserve monophyly two species previously assigned to Antanartia are transferred to Vanessa, Vanessa hippomenecomb.n. and Vanessa dimorphicacomb.n., resulting in a total of 22 species placed in Vanessa. A biogeographical analysis shows that in many cases the most geographically restricted species are sister to geographically widespread species, suggesting dispersal and allopatric speciation. Surprisingly, in almost all cases the divergences between widespread and restricted species are quite old (>5 Ma), suggesting long‐term isolation and stability of both vagile and sedentary species, despite the high (even intercontinental) vagility of many extant species and, by extension, ancestral species. The biogeography of Vanessa suggests that species vagility and allopatry do not fully explain the forces governing cladogenesis in this remarkable genus.