Contributions to Zoology, 70 (1) 23-39 (2001) SPB Academic Publishing bv, The Hague Hierarchical analysis of mtDNA variation and the use of mtDNA for isopod (Crustacea: Peracarida: Isopoda) systematics R. Wetzer Invertebrate Zoology, Crustacea, Natural History Museum of Los Angeles County, 900 Exposition Blvd., Los Angeles, CA 90007, USA, [email protected] Keywords: 12S rRNA, 16S rRNA, COI, mitochondrial DNA, isopod, Crustacea, molecular Abstract Transition/transversion bias 30 Discussion 32 Nucleotide composition 32 Carefully collected molecular data and rigorous analyses are Transition/transversionbias 34 revolutionizing today’s phylogenetic studies. Althoughmolecular Sequence divergences 34 data have been used to estimate various invertebrate phylogenies Rate variation across 34 lor lineages more than a decade,this is the first ofdifferent study survey Conclusions 35 of regions mitochondrial DNA in isopod crustaceans assessing Acknowledgements 36 sequence divergence and hence the usefulness ofthese regions References 36 to infer phylogeny at different hierarchical levels. 1 evaluate three loci fromthe mitochondrial ribosomal RNAs genome (two (12S, 16S) and oneprotein-coding (COI)) for their appropriateness in inferring isopod phylogeny at the suborder level and below. Introduction The patterns are similar for all three loci with the most speciose suborders ofisopods also having the most divergent mitochondrial The crustacean order Isopoda is important and nucleotide sequences. Recommendations for designing an or- because it has a broad dis- der- or interesting geographic suborder-level molecular study in previously unstudied groups of Crustacea tribution and is diverse. There would include: (1) collecting a minimum morphologically are of two-four species or genera thoughtto be most divergent, (2) more than 10,000 described marine, freshwater, sampling the across of interest as equally as possible in group and terrestrial species, ranging in length from 0.5 terms of taxonomic representation and the distributionofspecies, mm to 440 mm. are inhabitants of (3) They common several and surveying genes, (4) carrying out preliminary nearly all environments, and most are free- alignments, checking data for nucleotide bias, transition/ groups transversion ratios, and saturation levels before committing to living. Many are scavengers or grazers, although a large-scale sequencing effort. some are temporary orobligatory parasites of fishes and other crustaceans. Many species are shallow well water inhabitants, but some taxa are adapted Contents to life in the deep sea, subterranean groundwater, and thermal springs. Isopods are members of the Introduction 23 superorder Peracarida, and a synapomorphy of the Materials and methods 24 superorder is a brooding life style (there are no Sources of specimens and DNA preservation 24 larvae; is direct with DNA extraction, free-living development young primers, PCR amplification, and with the adult and thus there sequencing 26 emerging morphology) Sequence alignment strategy 26 is purported poor dispersal ability. Determining nucleotide composition, sequence The first isopod was described in 1764 (Asellus and divergence, transition/transversion bias 26 and the and taxo- GenBank .Geoffrey), group’s systematics submission 27 Present Results nomy has been bantered about ever since. 27 Nucleotide workers ten and the composition 27 recognize suborders, over past Sequence divergence 30 twenty years isopods have received considerable Downloaded from Brill.com10/08/2021 09:52:04AM via free access 24 R. Wetzer - mtDNA variation in isopods morphological systematic attention, with ordinal conserved than others (Cummings et al. 1995). Un- summaries provided by Bowman and Abele 1982, derstanding basic parameters such as patterns of Schram nucleotide substitution and variation Brusca and Iverson 1985, 1986, Wiigele rate among and Wilson sites is for of 1989, Brusca and 1991. Morphological important proper application DNA character-based, cladistic analyses have been carried sequence datato molecular systematic studies (Yang out for several isopod taxa (e.g., idoteid and arcturid 1994, Yang and Kumar 1996, Blouin et al. 1998, valviferans, Brusca 1984, Poore 1995; corallanid Whitfield and Cameron 1998). flabellifcrans, Delaney 1989; phreatoicids, Wiigele Mitochondrial 12S rRNA, 16S rRNA, and COI attractive 1989; janirid asellotans, Wilson 1994; serolids, genes are to crustacean evolutionary bio- Brandt 1988, 1992). logists because universal and crustacean-specific for Molecular techniques have invigorated crustacean primers are readily available polymerase chain the last dozen with reaction Saiki et al. and systematics over years primary (PCR; 1988) amplification, contributions stemming from higher-level system- amplified gene fragment sizes are amenable to atics. A comprehensive list of molecular phyloge- manual and automated sequencing techniques. Com- netic studies carried date for the known for these out to Crustacea parative arthropod sequences are the level and in Table and sufficient and at species higher appears 1. genes extracting adequate qual- No published studies exist within the Isopoda, ity DNA from ethanol-preserved specimens of although several mitochondrial (mt) DNA studies highly variable preservation are attainable goals considerable based on the I6S ribosomal RNA (rRNA) gene for organisms with a range in body in of Ph.D. size. of are progress (suborders Isopoda, Dreyer, By describing patterns sequence divergence dissertation, Ruhr-Universitat Bochum; species of within and among populations, species, genera, Thermosphaeroma, Davis et al., in review; and families, and suborders of isopods, the appropriate- Universite mitochondrial genera and families of Oniscidea, Michel, ness of three genes for evolutionary de levels be Poitiers). questions at various taxonomic can deter- As researchers turn to molecular methods, mined. mtDNA is being used to address both higher-level systematics and population-level questions. How- there when ever, are pitfalls using inappropriate Material and methods sequence data for phylogenetic inference. Selecting a genefor phylogenetic analysis requires matching Sources of specimens and DNA preservation the level of sequence variation to the desired taxo- nomic level Several have of study. recent papers The taxa used at each taxonomic (hierarchical) level focused the identificationof that useful shown on genes are of comparison are in Table 2. The suborder for at different taxonomic Flabellifera be phylogenetic analysis may not a monophyletic taxon levels (Brower and DeSallc 1994, Friedlander et (Kussakin 1979, Bruce 1981, Wagele 1989, Brusca al. Simon al. Sullivan 1994, Graybeal 1994, et 1994, and Wilson 1991), and relationships of the families 12S- and 16S rRNA et al. 1995). Mitochondrial included within the Flabellifera have also been genes and the protein-coding cytochrome oxidase controversial. In this study flabelliferan families c subunit I have been studied (COI) gene extensively are considered separate taxonomic entities and in within recently of (<5 diverged lineages arthropods figures are referred to by the family name followed million Sea urchins and butterflies years ago, mya). by “(Flabellifera).” Most specimens were collected exhibit similar rates for divergence a given gene, by the author; additional specimens were donated with the linear rate with time and 1.8-2.3% diver- by colleagues (see Acknowledgements). Most speci- million gence per years (Bermingham and Lessios mens were fixed and preserved in 95% ethanol, 1993, Brower 1994). However, when more ancient and in some instances DNA was extracted from (>75 of lineages mya) vertebrates are compared, specimens fixed in 70-75% ethanol. The latter different mtDNA with genes vary considerably specimens had body sizes >10 mm. respect to divergence rate, i.e., some genes are more Downloaded from Brill.com10/08/2021 09:52:04AM via free access Contributions to Zoology, 70 (1) - 2001 25 Table I. Molecular phylogenetic studies within Crustacea at the species level and higher with studies groupedby taxa. Genes studied include nuclear 18S rRNA, mitochondrial and 12S- 16S rRNAs, and protein-coding mitochondrial cytochrome oxidase c subunit I (COl) gene fragments. Taxon Hierarchical Reference Description Gene Level Crustacea class class Abele et al. 1989 Pentastomidia are Crustacea 18S18S rRNA Crustacea class Spears and Abele 1997 phylogeny of Crustacea 18S rRNA Crustacea class Spears and Abele 19991999 foliaceous limbs: Branchiopoda, Cephalocarida, and Phyllocarida I8S18S rRNA Branchiopoda class MannerBanner and Fugate 1997 phylogeny ofbranchiopods 12S rRNA Branchiopoda class SpearsSpears and Abele 2000 phylogeny ofbranchiopodsbranchiopods 18S rRNA Branchiopoda: Cladocera order Lehman etct al. 1995 phylogeny ofDaphnia 12S rRNA Branchiopoda: Cladocera subgenus/speciessubgenus/species Colbourne andand Hebert 1996 Daphnia 12S12S rRNA Branchiopoda: Cladocera speciesspecies Taylor et al.al, 1998 cryptic endemism of Daphnia 16S16S rRNA Maxillopoda class Abele et al. 1992 class relationships ISSrRNA18S rRNA Maxillopoda: Maxillopoda: Cirripedia suborder Spears et al. 1994 thecostracan relationships I8S rRNA Maxillopoda: Cirripedia speciesspecies vanSyocvanSyoc 1995 Pollicipes diversitydiversity COI Mizrahi of Maxillopoda:Maxillopoda; Cirripedia genus Mizrahi et al. 1998 phylogenetic position ofIbla 18S18S rRNA select thoracican Maxillopoda: Cirripedia family/genus Harris et al. 2000 barnacles 18S rRNA Maxillopoda: Perl-Treves