Arthropod Phylogenomics:
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Mitogenomic analyses of Arachnida using complete mt. genomes

In recent years, mitochondrial gene data has been extensively used to provide inferences about evolutionary history. The arachnids (spiders, scorpions and allies) are no exception, as most studies exploit small disparate fragments of the genomes for their data. In contrast, I am interested in the evolution and phylogenetic utility of the mitochondrial genome as a whole, focusing on understanding mutation accumulation biases, changes in sequence conservation and differential structural architecture.

This is my most recent research area, part of an NSF project led by Dr. Susan Masta and Dr. Jeff. Boore. The project ties in well with my broader research interests, focused around evaluating the systematic utility and molecular evolution using large-scale genetic sequence resources. As part of this project, we have sequenced mitochondrial genomes from a wide taxonomic sample of arachnids, including some obscure lineages (orders), like Ricinulei and Schizomida. I have been responsible for a mixture of lab. based and analytical aspects of the project, ranging from genome amplification and gene annotation to comparative molecular analyses.

Left: The complete mitochondrial
genome from the American horse-shoe crab Limulus polyphemus.

This link provides more about biology and anatomy of horse-shoe crabs (Xiphosura).

This mitochondrial genome was published by: Lavrov, Boore and Brown (2000).

More details about the status
of our results, and forthcoming arachnid mitochondrial genomes are available HERE.

 

We are have also included several Pycnogonida in our target taxa, which are considered by some to be allied chelicerates. The data generated by this project should provide insights on the relationships among the Arachnida, and help us better understand the processes of molecular evolution of arthropod mitochondrial genomes.

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Phylogenomic analyses of insect orders using ribosomal proteins

There are many outstanding systematic questions about holometabolan insects, i.e. those with a different ecology between larval and adults. Holometabolan insects generally have distinct pupae and undergo metamorphosis (Kristensen 1999; Beutal and Pohl 2006). However, one order - twisted-wing parasites (= Strepsiptera), are tetatively placed in the Holometabola, but are an enigma. Unlike typical Holometabola, they have an unusual development, lacking a distinct pupal stage [hypermetamorphosis]. Some larval strepsipterans also have external wing-buds, whereas holometabolan wings typically develop via an externalization of imaginal discs at metamorphosis. Along with many others, I am interested the phylogenetic placement of the enigmatic order Strepsiptera, which has vexed arthropod systematists for many, many years.

Specific areas of interest.

In my opinion, there has been (and still is) an over-reliance on rRNA in molecular phylogenetic studies of insects. The most productive research is focused on generating rRNA data over across a comprehensive taxon sampling (e.g. Whiting 2002a/b). This approach has certainly clarified several issues about the holometabolan phylogeny, but also raised other questions. During my doctoral research, I focused on understanding the molecular evolution and phylogenetic utility of proteins associated with ribosome, rather than rRNA. I am interested in the molecular processes driving co-evolution of multiple genes, especially for ribosomal proteins (RPs). My work has focused on elucidating the dominant factors that may cause shifts in rates of mutaton accumulation, and how such pressures can affect multiple genes scattered across the nuclear genome.

The figure above shows a molecular phylogeny based on 18S ribosomal RNA (rRNA) from Huelsenbeck 2001. These analyses show exceptionally long branches
in both Diptera and Strepsiptera, and hence the highly derived nature of their rRNA sequences. The big question is, are these long branches a result of convergence

or represent a synapomorphy?!?

Background on Insect Relationships

The bulk of Arthropod species diversity are insects, united by characters such as segmented tarsi and annulated antennae (Kristensen 1995). Relationships among major lineages of insects have been considered in treatises focused on the Apterygota (Bitsch and Bitch 2000), Palaeoptera (Hovmöller, Pape and Kalllersjö 2002), Polyneoptera (Whiting, Bradler and Maxwell 2003; Terry and Whiting 2005) and Holometabola (Kristensen 1995; 1999; Whiting et al.1997; Whiting 1998a/b; 2002a/b; Kjer 2004; Beutal and Pohl 2006). Together, these studies have led to some interesting conclusions, like disputing the monopyly of Mecoptera (Whiting 2002b), or the suggested re-evolution of wings in phasmids (Whiting, Bradler and Maxwell 2003). Gradually, the insect phylogeny is becoming clearer, allowing systematists to make polarized inferences about the evolutionary history of this huge radiation.

Origins of ususal molecular evolution

My main research involves looking at how best to use protein coding genes to provide inferences on insect phylogenetic relationships, particularly the Holometabola. However, by necessity, it is important to understand the processes that may generate different patterns of molecular evolution across taxa. There are several interesting aspects of strepsipteran biology that could affect their molecular evolution. For example, they show phenotypic adaptations to exploit several types of host insect (hence, what about molecular adaptions?), and they have unusual life-histories (larvae and females are endoparasites, while mature males are free-flying (and hence, how does this affect their population dynamics and gene flow?).

Why holometabolan insects?

Holometabolan insects provide a great set of taxa to evaluate the molecular evolution of the nuclear genome. Genetic studies of arthropods have focused on holometabolan insects, and there are complete nuclear genomes from flies (Drosophila spp. Anopheles gambiae, etc), a moth (Bombyx mori) and the honeybee (Apis mellifera). The bias towards holometabolan insects is also true of compilations of expressed sequence tag (EST) sequences. These are synthetic copies of mRNA sequences generated by reverse-transcription. I am also interested in understanding how best to use these sequence resources to generate large datasets of nuclear genes.

The Whiting lab. at BYU (USA) is one of the most productive for generating and evaluating phylogenetic data from insects. Here.

The Blaxter lab. in Edinburgh (UK) are also using compilations of EST data for phylogenetic analyses. Furthermore, compilations of published Arthropod EST data are now available. Here

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Comparative transcriptomic analysis of Coleoptera using EST sequences

Overview of Coleopteran EST research

One aim of my research with Coleoptera (beetles) is to evaluate the utility of expressed sequence tag data (ESTs) for clarifying relationships among taxa at the deepest hierarchical levels. For example, my research has focused on resolving relationships among major suborders and series. I am strongly in favor of generating molecular sequence data using an 'EST approach'. These techniques mean that development of gene/taxon specific primers is not necessary, and therefore allows rapid and comprehensive sampling of multiple gene sequences from taxa where little or no genomic data was known previously.

Together with co-authors, our research has demonstrated how the EST approach can allow systematists to quickly sample data from multiple genetic loci across lineages with broad taxonomic diversity. Some of this work has now been published with Dr. Joseph Hughes and Dr. Alfried Vogler. We first evaluated which genes are most commonly detected in arthropod EST libraries (Hughes et al. 2006), and demonstrated how best to exploit the recovered sequences to reconstruct phylogenetic relationships of the Coleoptera.

Background on Coleopteran Relationships

Morphological data strongly supports coleopteran monophyly (i.e. 28 synapomorpies from adults alone, Lawrence and Newton 1995; Beutal and Haas 2000). The order is dominated by two diverse suborders, Adephaga (~30,000 spp), and Polyphaga (over 300,000 spp), believed to reflect a deep-rooted phylogenetic cleavage (Crowson 1955; 1960). As a whole, the origins of the Coleoptera as a whole date to the lower Permian (Rohdendorf 1944). In the earliest fossils, elytral veination appears to be intermediate to the order Megaloptera and the small coleopteran sub-order Archostemata ([~20 spp] Crowson 1960; Lawrence, Slipinski and Pakaluk 1995). According to Crowson (1955; 1960), ancestral Coleoptera first diverged into three ‘stocks’, the wood-boring Archostemata, the predacious Adephaga and a group of mould-eating beetles that gave rise to the diverse Polyphaga. A fourth sub-order, Myxophaga was erected for anomalous forms (~50 spp), adapted to algae-feeding in moist or hygropetric habitats (Crowson 1960). Relationships among the four sub-orders of Coleoptera are controversial (see Lawrence and Newton 1995 for an nice overview).

Adapted from Fig. 3 in Hughes et al. 2006, and inferences on the relationships among the four beetle suborders (1) Archostemata (Micromalthus), (2) Adephaga (Cicindela, Carabus and Meladema), (3) Myxophaga (Sphaerius) and (4) Polyphaga (remaining spp). Note, ArchostEmata is mis-spelt.

Phylogenetic Analyses of Diverse Lineages.

A generally held view is that the diversity of taxa used in phylogenetic analyses should reflect the diversity of the lineage. However, acheiving this can be difficult when a lineage is hyper-diverse (i.e. species rich). The Coleoptera (beetles) are the most speciose insect order, with some 350,000 described species (Erwin 1982; Arnett 1985; Hammond 1992; Beutal and Haas 2000; Caterino et al. 2002; Whiting 2002a,b; Grimaldi and Engel. 2005). The morphological diversity of the Coleoptera limits the resolution that comparative anatomy can provide. Different beetle species are adapted to an array of terrestrial habitats as wells as freshwater, sometimes leading to extreme phenotypic differences. Diversity restricts morphological comparisons across the Coleoptera as a whole.

The Molecular Hyper-Diversity of Beetles.

I am interested in the diversity of the Coleoptera at the genetic level, especially in the evolution of their nuclear genes. For example, understanding how and why different taxa can display radically different nucleotide usage patterns, varying intron placements and heterogeneous genome structure. Clearly, it is important to clarify which taxa are the most suitable to be used in phylogenetic analyses, encompassing genetic diversity. In many cases rRNA data or mitochondrial genes have shown that certain taxa display unusual patterns of molecular evolution. I am interested in understanding the reasons why some lineages experience atypical molecular patterns, as the inclusion of such taxa often have important consequences for results of phylogenetic analyses.

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Conservation Genetics of Brachypelma tarantulas

Tarantula spiders (Araneae; Theraphosidae) are fascinating group of arthropods for diverse research questions. One aspect of my work on these large spiders is examining how individuals and populations are distinct at the genetic level. One genus, the Mesoamerican Brachypelma is the focus of my research. This group includes several species protected under international law (CITES, convention in trade in endangered species), as well as national laws in several countries. Members of this genus are protected due to extinction risks from over-collecting by the exotic pet trade, as well as habitat destruction.

Relating Threatened Lineages as Species to their Poulation Dynamics

I am interested in using phylogenetic information to understanding the evolutionary dynamics of this genus. First, genetic information can be used to focus conservation priorities among wild populations, determining genetic differences in situ across the present ranges of each species. I am interested in understanding gene flow, and how alternative molecular regions provide different insights. Second, genetic information can help ex situ captive breeding programs (source populations for reintroduction programs) to maintain biodiversity. Here, it is fundamental that captive breeding programs limit the potential risk of hybridization. Captive populations can be used to infer why only some populations of the threatened species still occur in the wild, and how best to ensure their future persistence.

Developing Non-Lethal Techniques and Tissue Sources

I am also interested in using the genus Brachyplema to evaluate non-lethal DNA sampling methods for genetic studies. Most studies with arthropods use preserved specimens as the source tissue. In current practices, the specimen acts as a voucher for others to examine later. While vouchers are critical for morphological studies, long-term maintenance of 'voucher DNA' is not possible, only in silico. I am interested in the development of genetic sequence resources from tarantulas as sets of reference markers, in a multigene extension of a barcoding approach. Genetic resources of reference DNA will be useful to national wildlife enforcement authorities, enabling them to quickly and efficiently determine the origin of spiders seized after an (alleged) illegal event.

Some great long-term ecological work is being conducted by Steve Reichling and collaborators on the theraphosid (and associated) arachnofauna of Belize. Other researchers are also interested the development of genetic sequence resources for tarantulas for wildlife law enforcement, and development of non-lethal methods to sample DNA, i.e. from exuviae (e.g. Peterson et al. 2006).

A great site to learn more about spiders in the genus Brachypelma can be found here: http://www.brachypelmas.co.uk/
I recommend that everyone enjoys using the words "genus AND Brachypelma" together while they can,
as these words won't stay affiliated together much longer.

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Now, Some Wise Words:

  "Time flies like an arrow, but fruit flies like a banana."

   Groucho Marx.

Then, a warning to systematists who ignore non-lethal DNA sampling.

   "He that breaks a thing to find out what it is.... has left the path of wisdom."

   Gandalf the Gray.

     (From: The Lord of the Rings; The Fellowship of the Ring [John Ronald Reuel Tolkien])

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REFERENCES             Back to Arachnids          Back to Insects          Back to Coleoptera          Back to Tarantulas

Arnett, R. H. 1985. General considerations. In American Insects: A Handbook of the Insects of America North of Mexico. Van Nostrand Reinhold, New York.

Beutal, R. G. and F. Haas. 2000. Phylogenetic relationships of the suborders of Coleoptera (Insecta). Cladistics 16:103-141.

Beutel, R. G. and H. Pohl. 2006. Endopterygote systematics – where do we stand and what is the goal (Hexapoda, Arthropoda)? Systematic Entomology 31(2):202-219

Bitsch, C., and J. Bitch. 2000. The phylogenetic interrelationships of the higher taxa of apterygote hexapods. Zoological Scripta 29:131-156.

Carmean, D. and B. J. Crespi. 1995. Do long branches attrach flies? Nature 373:666.

Caterino, M., V. L. Shull, P. M. Hammond, and A. P. Vogler. 2002. Basal relationships of Coleoptera inferred from 18S rDNA sequences. Zool. Scripta 31:41-49.

Crowson, R. A. 1955. The natural classification of the families of the Coleoptera. Nathaniel Lloyd, London.

Crowson, R. A. 1960. The phylogeny of Coleoptera. Ann. Rev. Entom. 5:111-134.

Erwin, T. L. 1982. Tropical forests: their richness in Coleoptera and other arthropod species. Coleopt. Bulletin. 36:74-75.

Grimaldi, D. and M. S. Engel. 2005. Section 10. Coleoptera: early fossils and overview of past diversity; Archostemata; Adephaga; Myxophaga; Polyphaga; Strepsiptera: the enigmatic order. In Evolution of the Insects. Cambridge University Press.

Hovmöller, R., T. Pape and M. Kalllersjö. 2002. The Palaeoptera problem: Basal pterygote phylogeny inferred from 18S and 28S rDNA sequences. Cladistics 18:313-323.

Hammond, P. M. 1992. Species inventory. In Global Biodiversity, Status of the Earth’s Living Resources. Groombridge, B. Ed. Chapman and Hall, London. Pp. 17-39.

Hughes, J., S. J. Longhorn, A. Papadopoulou, K. Theodorides, A. De-Riva, M. Mejia-Chang, P. G. Foster, and A. P. Vogler. 2006. Dense taxonomic EST sampling and its applications for molecular systematics of the Coleoptera (beetles). Mol. Biol. Evol. 23:268 - 278.

Huelsenbeck, J. P. 2001. A Bayesian perspective on the Strepsiptera problem. Tijdschrift voor Entomologie. 144:165-178.

Huelsenbeck, J. P. 1997. Is the Felsenstein zone a fly trap? Syst. Biol. 47:519-537.

Kjer, K. M. 2004. Aligned 18S and insect phylogeny. Syst. Biol. 53: 506-514.

Kristensen, N. P. 1995. Forty years’ insect phylogenetics. Hennig’s “Kritische Bemerkungen” and subsequent developments. Zool. Beitr. N.F. 36:83-124.

Kristensen, N. P. 1999. Phylogeny of endopterygote insects, the most successful lineage of living organisms. Eur. J. Entom. 96:237-253.

Kukalová-Peck, J. and J. F. Lawrence. 1993. Evolution of the hind wing in Coleoptera. Can. Entom. 125:181-258.

Kukalová-Peck, J. & Lawrence, J. F. 2004 Use of hind wing characters in assessing relationships among coleopteran Suborders and major endoneopteran lineages. European Journal of Entomology 101:95–144.

Lawrence, J. F., S. A. Slipinski, J. Pakaluk. 1995. From Latreille to Crowson: a history of the higher level classification of beetles. In Biology, phylogeny and classification of Coleoptera: Papers celebrating the 80th birthday of Roy A. Crowson. Palaluk, J. and S. A. Slipinski. Eds.. Muzeum I Instytut Zoologii PAN. Warszawa. Pp. 87-154.

Lawrence, J. F. and A. F. Newton Jr. 1995. Families and Subfamilies of Coleoptera (with selected genera, notes, references and data on family-group names). In Biology, Phylogeny and classification of Coleoptera: Papers celebrating the 80th birthday of Roy A. Crowson. Palaluk, J. and S. A. Slipinski. Eds. Muzeum I Instytut Zoologii PAN. Warszawa. Pp. 779-913.

Peterson, S. D., T. Mason, S. Akber, R. West, B. White and P. Wilson. 2006. Species identification of tarantulas using exuviae for wildlife law enforcement. Conserv. Genetics. DOI 10.1007/s10592-006-9173-2.

Pix, W. G. Nalbach, and J. Zel. 1993. Strepsipteran forewings are haltare-like organs of equilibrium. Naturwissenschaften 80:371-374.

Rohdendorf, B. B. 1944. A new family of beetles from the Permian of Urals. Doklady Akademia Nauk SSSR 44:277-279.

Terry, M. D. and M. F. Whiting. 2005. Mantophasmatodea and Phylogeny of the Lower Neopterous Insects. Cladistics. 21: 240-257.

Wheeler, W. C., M. Whiting, Q. D. Wheeler, and J. M. Carpenter. 2001. The phylogeny of the extant hexapod orders. Cladistics. 17:113-169.

Whiting, M. F. and Wheeler, W. C. 1994. Insect homeotic transformation. Nature 368:696.

Whiting, M. F., J. C. Carpenter, Q. D. Wheeler and W. C. Wheeler 1997. The Strepsiptera problem: Phylogeny of the holometabolous insect orders inferred from 18S and 28S ribosomal DNA sequences and morphology. Syst. Biol. 46:1-68.

Whiting M. F. 1998. Long-branch distraction and the Strepsiptera. Syst. Biol. 47:134-138.

Whiting, M. F. 1998. Phylogenetic position of the Strepsiptera: review of molecular and morphological evidence. International Journal of Insect Morphology and Embryology 27:53-60.

Whiting M. F. 2002a. Phylogeny of the holometabolous insect orders: molecular evidence. Zool. Scripta. 31:3-15.

Whiting, M. F. 2002b. Phylogeny of the holometabolous insect orders based on 18S ribosomal DNA: when bad things happen to good data. In Molecular Systematics and Evolution: Theory and Practice. DeSalle, R., G. Giribet and W. Wheeler (Eds). Birkhäuser, Velag/Switzerland.

Whiting, M. F., S. Bradler and T. Maxwell. 2003. Loss and recovery of wings in stick insects. Nature. 421:264-267.

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