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 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 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 et al. 2000 thecostracans: Verruca, Cirripedia genus Verruca, Paralepas,
and Dendrogaster 18SISSrRNArRNA Maxillopoda;Maxillopoda: Copepoda species Bucklin et al. 1992 intraspecific and interspecific
patterns (Calanoida) 16S rRNA Maxillopoda: Copepoda speciesspecies Bucklin et al. 1995 species of Calanus (Caianoida)("Calanoida) 16S rRNA Malacostraca: Decapoda order Kim and Abele 1990 ordinal relationshipsrelationships ISSrRNA18S rRNA Malacostraca: Decapoda order Abele 1991 morphology and molecular data 18S rRNA
Malacostraca: of Brachyura infraorder Spears et al. 1992 monophyly of brachyuran crabs 18S rRNA
Malacostraca: of Brachyura family Schubart et al. 2000a phylogeny brachyuran families I6S16S rRNA Malacostraca: Anomura infraorder Cunningham et al. 1992 king crabscrabs and hermit crabs 16S rRNA Malacostraca: Crandall et al. 19951995 Australian 16S rRNA Decapoda genusgenus Crandall crayfish (Parastacidae)
Malacostraca: Lawler and Crandall 1998 Euastacus and Decapoda genusgenus Astacopsis 16S rRNA Malacostraca: Decapoda species Ponniah andand Hughes 1998 Euastacus relationships 16S rRNA
(Parastacidae) Malacostraca: Decapoda subgenus Crandall and Fitzpatrick 1996 crayfish (Cambaridae) 16SrRNA16S rRNA Malacostraca: Decapoda species Crandall 1998 Ozark crayfishes (Cambaridae) 16S rRNA Malacostraca: Decapoda species Tam et al. 1996 divergence and zoogeography of
mole crabs mole crabs (Hippidae) 16S rRNA Malacostraca: Decapoda genus/subgenus Sturmbauer et al. 1996 fiddler crabs (Ocypodidae) 16S rRNA Malacostraca: Decapoda species Geller et al. 1997 cryptic invasioninvasion of Carcinus
(Carcinidae) 16SrRNA16S rRNA Malacostraca: Tam and Kornfield 1998 ofclawed lobsterslobsters Decapoda genus phylogeny
(Nephropidae) 16S rRNA Malacostraca: Decapoda species/subspecies Sarver et al.al. 1998 species/subspecies differentiation
of Panulirus argus (Palinuridae) 16S rRNA Malacostraca: Euphausiacea species Patarnello et al. 1996 relationshipsrelationships ofkrill 16S rRNA
a Malacostraca:acostraca: al,al. variation in crabs Brachyura species Schneider-Boussard et sequencesequence stone
1998 Menippe adina and M. mercernaria 16S rRNArRNA Malacostraca:a acostraca: Brachyura subfamily/genus Kitaura et al.al, 1998 relationshipsrelationships of Ocypodidae 12S rRNArRNA and 16SI6S rRNA
Malacostraca: of Decapoda species -< Schubart et al. 1998 species Sesarma (Grapsidae) 16S rRNArRNA Malacostraca: Decapoda species Schubart etet al. 1998 JamaicanJamaican grapsid crabs (Grapsidae) 16S rRNArRNA and COI Malacostraca: Decapoda subfamily/genus Schubart etet al. 2000b phylogenyphylogeny of Grapsoidea 16S rRNArRNA Malacostraca:a acostraca: Amphipoda genus/species FranceFrance and Kocher 1996 deepsea Lysianassidae I6S16S rRNA via Malacostraca:acostraca: Mysidacea family Casanova, J.-P.J.-P. etet al. 1998 LophogastridaLophogastrida !6SrRNAI6S rRNA Malacostraca:a acostraca: Isopoda genus/species Michel-Salzat and Bouchon phylogenetic relationship
2000 oniscids i amongamong 16SrRNA16S rRNA M Malacostraca:aiacostraca: Isopoda genus/species Held 2000 phylogeny and biogeography of I6S16S rRNArRNA
serolidsserolids and 18S18S
rRNA 26 R. Wetzer - mtDNA variation in isopods
DNA extraction, primers, PCR amplification, and strands were sequenced on an ABI 377 automated
sequencing sequencer. Nucleotide sequences were edited us-
ing the Sequencher software package (ver. 3.1,
Debris and shaken off GeneCodes Ann and ectoparasites were specimens Corp., Arbor, MI), sequences
by submerging them in deionized water and ex- were searched for similarity to other arthropods
posing them to ultrasound waves for 5-10 seconds. using BLAST (Basic Local Alignment Search Tool,
Specimens were then rinsed 3-4 times in deion- URL: http://www.ncbi.nlm.nih.gov/BLAST/index.
ized Since in the ofCOI water. isopods vary considerably body html). Additionally, accuracy sequences
size, two different extraction protocols were used. was verified by translating nucleotides to amino
DNA from specimens less than 3 mm in length acids with MacClade 3.06(Maddison and Maddison
extracted standard and all verified for was using a phenol-chloroform 1996), sequences were proper
protocol (Cunningham and Buss 1993). Appendages reading frame.
(antennae, pereopods, or pleopods) were dissected off specimens larger than 5 mm and tissue simi-
25 larly extracted. Alternatively, mg of tissue (en- Sequence alignment strategy
tire specimen, anterior, or posterior half of speci-
extracted the Tissue Kit men) were using QIAamp Thirty-three COI sequences were aligned by hand
One four of (Qiagen, Inc., Valencia, CA). to pi since there were no insertions or deletionsof nucleo-
DNA in PCR template were used 50-pl reactions. tides or amino acids. One data set was prepared
The COI the sequence was amplified using for amino acid and a second data set for nucleotide
Folmer et al. universal The (1994) primers (LCOI490 analyses. multiple-sequence-alignment program
-442 and HC02I98, base pairs, bp), and Palumbi CLUSTALW 1.74 (Gibson et al. 1996) was set to
et al. universal 16Sar and !6Sbr = (1991) primers default settings (slow/accurate gap open penalty
were used for the 16S rRNA = fragment (-378 bp). 15.00, gap extension penalty 6.66, /:-tuple size
A -275 the 12S rRNA = transitions and used bp region of gene was am- 2, not weighted) to align 49
18 rRNA plified using peracarid specific primers (I2SCRF: and 16S and 12S rRNA sequences, re-
5'-GAG ACT GAC GGG CGA TAT GT-3'; spectively.
12SCRR: 5'-AAA CCA GGA TTA GAT ACC CTA
TTA T-3'),
For the PCR reaction, Perkin Elmer (Foster City, nucleotide Determining composition, sequence 10X buffer and CA) or Promega (Madison, WI) divergence, and transition/transversion bias the manufacturer’s respective Taq DNA polymerase
(2.5 units) were used with an initial denaturation The essential component of genomic structure are of period 3 minutes at 95°C, followed by 35 cycles the two linear polynucleotide chains composed of at 94°C for 15 seconds and extension for 1.5 minutes two purines (adenine [A] and guanine [G]) that at 72°C. from 48°C Annealing temperatures ranged hydrogen-bond to two pyrimidines (thymine [T]
(COI) to 52°C rRNA and 16S for I (12S rRNA) and cytosine [C], respectively). Nucleotide fre- minute. PCR (3-6 ) was amplification product pi quency can result in taxon- and gene-specific pat- electrophoresed an bromide- through ethidium terns of nucleotide composition. The phylogenetic stained 1-2% and the product was agarose gel, analysis program PAUP* version 4.062 (Swofford checked for size. PCR proper Remaining product 1999) was used to determine nucleotide composi- was with purified polyethylene glycol (PEG) or tion. with G-50 Sephadex (Sigma Chemical, Inc.) and A method of summarizing the relationship be-
Centrisep columns (Princeton Adel- tween two is their fraction Separations, sequences by (or per- phia, NJ), or if the necessary, gel purified using centage) ofsimilarity or dissimilarity. In its simplest Gel Purification Qiagen Kit. DNA was then cycle form, the similarity is equal to the numberof aligned with ABI sequenced (Applied Inc., identical Biosystems sequence positions containing residues
Foster City, CA) and both Big Dye terminators, (bases or amino acids) divided by the number of Contributions to Zoology, 70 (I) - 2001 27
Lironeca vulgaris (Cymothoidae), the data are from
two ovigerous females from which two individual
young (mancas) were removedand for this analysis
treated as “individuals.”
Transition/transversion tables (ti/tv) were gen-
erated from the same data sets as above with PAUP*
and plotted with a program written by N. D.
Pentcheff (unpublished) (Fig. 5). The number of
transitions versus the number of transversions in
all pairwise comparisons of COI, 16S rRNA, and
12S rRNA are corrected for sequences sequence
length variation by dividing the ti/tv ratio by the
number of nucleotides in each sequence.
GenBank submission
GenBank accession numbers for sequences listed F'g- I Percent . divergence (“uncorrected p”) for cytochrome follows: in Table2 are as COI sequences AF255775- oxidase c subunit 1 (147 amino acids, 33 taxa) plotted against AF255791/AF260834-AF260846, 16S rRNA se- taxonomic level. Minimum and maximum divergence measure AF260847- quences AF259531-AF259547, expressed as range (bar). Number of pairwise comparisons indicated in and 12S rRNA AF259521- parentheses on right. AF260870, sequences
AF259530, AF260558-AF260562, AF260564.
sequence positions being compared. Dissimilarity
is merely the proportion (p) of nucleotide sites (n)
at which the Results two sequences being compared are different = /n. if (n ), p n For example, two taxa d d are Nucleotide represented by 20 aligned nucleotides and they composition differ at 5 sites, the “uncorrected p” value is 0.25.
These two Nucleotide of 16S and sequences can also be said to have 75% composition COI, rRNA,
similarity. 12S rRNA sequences are provided in Table 3. COI
nucleotide is for all three Percent-sequence divergence (“uncorrectcd p”) composition reported values in codon first and second obtained using PAUP* arc summarized positions: positions only, Figs. 1-4 for COI amino acids, COI nucleotides, and third positions alone. The G statistic for the 1 6S rRNA, and 12S rRNA within individuals, log-likelihood ratio goodness-of-fit test was used species, genera,families, and suborders. Sequence to determine whether nucleotide composition was
comparisons a A < were made at the lowest taxonomic equal within given gene. nucleotide bias (p 'cvel possible. For example, for COI amino acids 0.001) was found for all genes. For COI (all posi- (Fig. 1), the bias. two sphaeromatids Sphaeramene and tions) there is roughly a 7% A+T The A+T
Sphaeroma when third were bias are compared at the genus level. Since nearly disappears positions
only one species in each removed, Ts are favored over As. COI third genus was sequenced, this yet was the 68% Both ribosomal only comparison possible at this hierar- positions alone are ca. A+T. chial level. Plots in all instances RNA have A+T represent sequences genes nearly equal composition of individuals from 58% and 62% A+T for 16S- and 12S a single specimen lot, i.e., a (ca. rRNA, single population, except Caecidotea (Asellota) respectively). which comes from two populations a few kilometers
‘'part. All other collection localities ranged in area bom 0.5 to 3 nr. In the case of the fish ectoparasite 28 R. Wetzer - mtDNA variation in isopods
Sur, Wales, Park. Creek MBRD Diego. D.C., Luderitz. Park.Park. Rock SIO Cumberland San South National Carolina, California Angeles. Carolina, of material. Tasmania. Western Perth. south New Baha Los south of NationalNational Washington, Maryland, California, Georgia,Georgia, California,California, Creek South de South Australia, Australia, table. Columbia.Columbia. Charleston.Charleston. sources Locality Australia, Australia, BoydsBoyds Australia, Australia, USA, Rock USA, Park. USA, wetwet USA, Mexico, Bahia USA,USA, Island. USA, USA, Namibia, and sequenced, rRNA AF259525 AF260564 AF259524 AF259523 AF259529 AF259522 AF259527 genes I2S12S for numbers rRNArRNA submittedsubmitted submited AF259531 AF260869 AF259532 AF260870 AF259533 AF259534 AF260858 AF260859AF260859 AF260860 AF260847 AF259535 AF260865 AF260861 AF260863 AF260862 AF259536 AF260853 AF259537 accession 16S not not GenBank numbers, Genes COI AF255775 AF255776 AF255777 AF255778 AF260834 AF260835 AF260836 AF260837 AF260837 AF255779 AF255780 AF255781 reference Reference No. 288/357 authors’ 398 328 393393 286 345 329 389 181 184 199 207 380 381 173 382 383 347 390390 384 195 387 196 219 180 290 394394 taxonomy, Species thompson thompsoni buntiae buntiae buntiae buntiae palustris palustris epilittoralis epilittoralisepilittoralis epilittoralis epilittoralisepilittoralis dubia dubia dubia vulgare vulgare punctatus exoticaexotica exoticaexotica occidentalis occidentalis lichtensteini lichtensteini lichtensteini current sp. sp. analyses, in examined Colubotelson ColubotelsonColubotehon Paramphisopus Paramphisopus Armadillidium ArmadillidiumArmadillidium Genus Crenoicus Crenoicus Crenoicus Crenoicus Caecidotea Caecidotea Ianiropsislaniropsis Ianiropsislaniropsis Ianiropsislaniropsis Ianiropsislaniropsis Joeropsis Joeropsis JoeropsisJoeropsis Tylos Ligia Ligia LigiaLigia LigiaLigia Glyptoidotea GlyptoidoteaGlyptoidotea GlyptoidoteaGlyptoidotea taxa Isopod 2. Suborder/FamilySuborder/Family Phreatoicidae Asellidae Joeropsidae Armadillidiidae Tylidae Ligiidae Table Phreatoicidea Asellota Janiridae Oniscidea Valvifera Idoteidae - Contributions to Zoology, 70 (I) 2001 29
side Johns end Sur, of DiegoDiego Monterey west Liideritz. Port north Liideritz.Liideritz, Monterey Liideritz. Liideritz Monterey San of Diego of of of of California Angeles.Angeles. Island. Carolina, University Island, Carolina, Island. Carolina, Harbor. south Victoria, southsouth Tasmania, Peninsula. south San Harbor. British Columbia. south south Los California,California, Washington, Bay.Bay. Washington, South California, California,California, BajaBaja South South de South Whidbey Whidbey de USA,USA, Bay. USA, Namibia, USA, Island. Australia, Philips Canada, Columbia, British Namibia, USA, Pritchard’s Australia, Tasmania Peninsula. Namibia, Namibia, County. Mexico, California, County. CharlestonCharleston of British USA, of USA, USA, USA,USA, BahiaBahia USA,
submitted AF259526AF259526 AF260560 AF260561 AF260562 AF259528AF259528 AF259521 AF260558 AF260559 AF259530 not
submitted submitted submitted AF259538 AF260854 AF260855 AF260856 AF260857 AF259539 AF259545 AF260866 AF260867 AF260868 AF259540 AF259541 AF260864 AF259542 AF259543 AF259544 AF260848 AF260849AF260849 AF260850 AF260851 AF260852 AF259546 AF259547 not not not
AF255782 AF255783 AF255789 AF260845 AF255784 AF260846 AF255785 AF255786 AF260838AF260838 AF255787 AF255788 AF260839 AF260840 AF260841 AF260842 AF260843 AF255790 AF259547 AF260869
,
182 420 354 363 363 395 215 331331 358 386 335 400 324 360 391 183 216 284 392 287 336 349 169 210 289 403 179 330 388 198 211211 402 385 200 218 401 213 396
wosnesenskii resecata resecata wosnesenskiiwosnesenskii wosnesenskii ungulata laticauda laticauda laticauda oregonensis oregonensis oregonensis polytylotos polytylotos quadridentata quadridentata bakeri harfordi harfordi harfordi harfordi rugicaudarugicauda rugicauda rugicauda chillonichiltoni chiltoni chiltoni mayanamayana vulgaris vulgaris vulgaris praegustator praegustator sp. sp. bakeribakeri bakeri
Gnorimosphaeroma t Idotea Idotea Idotea Idotea Idotea Paridotea Synidotea Synidotea Synidotea ApanthuraApanthura Apanthura Gnorimosphaeroma Gnorimosphaeroma Gnorimosphaeroma Sphaeramene Sphaeramene Sphaeroma SphaeromaSphaeroma SerolinaSerolina Serolina Serolina Cirolana Cirolana Cirolana Cirolana Cirolana Cirolana Cirolana Excirolana ExcirolanaExcirolana Excirolana Excirolana Lironeca Lironeca LironecaLironeca OlenciraOlencira Olencira
» Anthuridea Anthuridae Flabellifera Sphaeromatidae Serolidae Cirolanidae Cirolanidae Cyraothoidae 30 R. Wetzer - mlDNA variation in isopods
The 33 in the Table 3. Nucleotide composition and sequence length (total same taxa were used nucleotide number ofnucleotides) for all isopods surveyed. A = adenine, comparisons (Fig. 2). Overall, these comparisons T = thymine, C = cytosine, G = guanine. Numeric values in were comparable to aminoacid divergence patterns. codon included parentheses following COl represent positions Here values for comparisons ofindividuals ranged in calculations. Values for nucleotides expressed as percent of from 15.8- total. The log-likelihood ratio goodness-of-fit test (G stat) was 0-3.2%, species 32.9-34.9%, genera
< df = suborders significant for all gene sequences (p 0.001), 3. 37.6%, families 22.9-34.5%, and across
20.7-35.5%. Gene A T C G Total No. The 16S rRNA data set 3) contained 49 Nucleo- G (Fig. sequence stat for 429 tides taxa which aligned bases were compared.
Among individuals, pairwise sequence divergences COI (1,2,3) 23.6 37.3 19.5 19,6 442 1141 ranged from 0-2.7%. Specimens of Oniscidea (Ligia COI (1,2) 19.6 35.1 22.4 22.9 295 521 exotica) from South Carolina and Georgia COI (3) 31.6 41.5 13.7 13.2 147 1142 popu- ~240 the 16S rRNA 35.8 29.9 16.3 18.0 378 1997 lations were separated by km, and se-
12S rRNA 32.8 32.7 14.8 19.7 275 521 quences obtained are identical. Sequence diver-
and cirolanid gences for oniscid, valviferan, species
comparisons ranged from 14.5-21.3%. Across-
for Sequence divergence genera divergences phreatoicids, asellotans,
and valviferans, sphaeromatids, cirolanids, cymo-
thoids from 8.9-49.1%. Of these Figures 1-4 summarize the pairwise sequence di- ranged compari- the Cirolanidae exhibit the diver- vergence for COI amino acids and nucleotides, and sons, greatest
16S- and These data (38.9-49.1%). Familial 12S rRNA sequences. are gences comparisons ranged arranged in taxonomic (hierarchical) fashion with from 34.4-49.1%. The subordinal comparisons were
28.2-49.1%. minimum and maximum sequence divergences observed for each taxonomic ranking. The 12S rRNA data set (Fig. 4) contained 18
taxa for which 312 bases used in The COI comparisons for 33 taxa are shown in aligned were
2. data 442 Three of individuals from Figs. 1 and This set is based on bases, comparisons. comparisons
were The i.e., 147 amino acids. Note all sequences were trun- single populations possible. two phreatoi-
cid were the valviferan cated to the length of the shortest sequence to elimi- sequences identical, two nate were 4.3% and the six spurious values due to unequal sequence length. sequences dissimilar, cirolanid 47-52% dis- Figure 1 summarizes the amino acid comparisons. sequence comparisons were
For similar. The trend toward di- individuals from the same population pairwise increasing sequence
is maintained for taxonomic levels from sequence divergence ranged from 0-4.1%. Phreatoi- vergences cid to with the sequences were identical, and comparisons of species to genera tofamilies suborder,
Asellota from value 50.2%. ranged 0.7-2.0%. Comparisons among greatest reaching
from individuals populations of Flabellifera are reported separately (see Methods, for discussion of bias taxonomic treatment of Flabellifera taxa) for Transition/transversion the members of the family Cirolanidae (1.4-4.1%) and used of substitutions Cymothoidae (0-1.4%). Comparisons of spe- A frequently measure is the cies of the calculation of transversions flabelliferan family Cirolanidae were transitions (ti) and (tv). observed to A have 13.6-14.7% sequence divergences. Transitions are substitutions between and G
At the genus level, five hierarchical comparisons (purines) or between C and T (pyrimidines). Trans- could be made with values ranging between 9.5- versions are substitutions between a purine and a 32% for all comparisons. At the family level, three pyrimidine. Generally, transitions occur more fre- hierarchical could be made with the than for comparisons quently transversions, even though any flabelliferan families exhibiting the largest range, given nucleotide position twice as many possible
17.7-33.6%. The largest range was clearly exhibited transversions may occur as transitions. Figure 5 the flabelliferan by family Sphaeromatidae. illustrates the transition/transversion (ti/tv) values Contributions to Zoology, 70 (I) - 2001 31
F‘S- 2. Percent for sequence divergence (“uncorrected p”)
cytochrome oxidase c subunit I (442 bases, 33 taxa) plotted
against taxonomic level. Minimum and maximum divergence
measure Number of expressed as range (bar). pairwise compari-
sons indicated in parentheses on right.
Fig. 3. Percent sequence divergence (“uncorrected p”) for 16S corrected for sequence length for each hierarchical rRNA (429 aligned bases, 49 taxa) plotted against taxonomic comparison individual, and ( species, genus,family, level. Minimum and maximum divergence measure expressed
Number of in suborder) as discussed previously. During the prepa- as range (bar). pairwise comparisons indicated
parentheses on right. ration of Fig. 5, the importance of correcting ti/tv ratios for sequence length became obvious when
stud- comparing multiple genes, and although some
ies have depicted ti/tv plots with regression lines, the non-independence of transitions and transver-
SI °ns make such depictions inappropriate (Purvis and Bromham 1997).
Excluding third codon positions (Fig. 5f-j)' re- duces ti/tv ratios (compare Fig. 5a-e to 5f-j). These results suggest that for suborder-, family-, and pos- S| bly gem/s-level comparisons, the third positions
arc saturated. In the ti/tv ratio comparisons for
individuals, and species, genera (Fig. 5a, f, k, 1, m > P, q, and r), the data points fall into roughly two clusters. This is in part the result ofthe num-
er an d kinds of taxonomic comparisons possible with this data set and reflects the larger divergences observed for the flabelliferan families Cirolanidae 4. Percent 12S and Fig. sequence divergence (“uncorrected p”) for Sphaeromatidae relative to all other isopods. rRNA (312 aligned bases, 18 taxa) plotted against taxonomic
'level. Minimum and maximum divergence measure expressed
I C>te scc l u cnce identical in 5a-e and Number of indicated in comparisons are Figure as range (bar). pairwise comparisons <- |
parentheses on right. 32 R. Wetzer ~~ mtDNA variation in isopods
For example, the data clouds in the bottom left of lation between the degree of amino acid bias and
and thus Fig. 5a, f, k, p represent Phreatoicidea, protein sequence composition impacting
Valvifera, Asellota, and Cymothoidae comparisons, molecular evolution of proteins and resulting in
clouds in the for the of whereas figure’s upper right are important implications interpretation
Cirolanidae and Sphaeromatidae. Similarly, the protein-based molecular phylogenies.
and comparisons for species and genera for 16S- The hypothesis of equal base frequencies (Table
is for these data. exhibits I2S rRNA (Fig. 51, m, q, and r) each form two 3) rejected Isopod mtDNA
AT clusters. Figure 51 shows comparisons for a high proportion of in both of the ribosomal valviferans (bottom left) and cirolanids (upper genesstudied. COI third codon positions have simi- right). In Fig. 5q the bottom left corner are lar AT proportions to the ribosomal genes, whereas phreatoicids, middle are valviferans, and the two COI first and second codon positions are only T-
The cloud the upper-right data points are cirolanids. rich, i.e., 35.1%. Altering subsets of isopod
in the bottom left of Fig. 5m are valviferans and taxa included in comparisons of base frequency
in the cymothoids, the larger cloud centerand upper among genes has no effect. Similarly, excluding right are cirolanids, sphaeromatids, and asellotans. highly variable regions of the 16S- and 12S rRNA
In the four data in the lower left where be Fig. 5r points sequences (i.e., regions alignment may
of the has portion figure are phreatoicids with ambiguous) no effect on nucleotide composition
valviferans in the center of the figure. In the fam- (data are not shown).
ily and suborder comparison for 16S-and 12S rRNA Overall these data suggest that isopod crustaceans
examined here have smaller bias (Fig. 5n, o, s, and t) generally more transversions a AT compared
occur than transitions, resulting from these genes’ to insects. Still, isopod AT bias is more similar to
AT bias and indicating that the data are saturated insects and other molting organisms (Ecdysozoa)
(i.e., increasing homoplasy masks phylogenetic (e.g., nematodes, Blouin et al. 1998) than to humans.
signal) at these hierarchical levels. In Fig. 5t, the An artifact of AT-rich mtDNA is that taxa have a
lone data left is in based point at the bottom a comparison tendency to group phylogenetic analyses of a cirolanid to an oniscid, clearly two distant taxa more on shared nucleotide composition than on whose low ti/tv value reflects saturation. These shared history (Flasegawa et al. 1993, Steel et al.
findings have broad implications for isopod phy- 1993), underscoring the importance of appropriate
logenetic studies, not only at a variety of hierar- substitution models when estimating phylogenetic chical but also for taxonomic The herein levels, specific groups. relationships. findings are congruent
with the 16S rRNA data for Australian freshwater
and Crandall which crayfish genera (Lawler 1998),
Discussion also found an AT bias; A=32.2%, T=35.3%,
C=10.8%, and G=21.7%. Similarly, Flanner and
Nucleotide composition Fugate’s (1997) study found the 12S rRNA gene
of orders have branchiopod crustacean to an average
Nucleotide of mitochondrial AT bias of A=34.3% and T=31.9%. The composition genomes findings varies For of Funk et al. for beetles re- among animal taxa. example, the com- (1995) phytophagus
mitochondrial vealed a AT bias to plete DNA sequence of Drosophila not only stronger compared
in has an AT content between 74-80% (see Clary and what has been found crustaceans, but the AT
Wolstcnholme bias for the 16S rRNA than for 1985), the honeybee is 84.9% AT was greater gene
(Croizer and Croizer 1993), whereas in humans it the COI gene (AT bias for 16S rRNA: A=37.3%,
is 55.5% AT (Anderson et al. 1981). Compositional T=41.2%; COI: A=28.9%, T=37.1 %). Whitfield and differences Cameron’s of taxa among homologous sequences have (1998) study hymenopteran been attributed exhibited the of AT nucleotides to both variation in selective con- greatest proportions straints and of measured. For the 16S rRNA changes in mutation patterns during any organism yet
A+T evolutionary divergence (Pcrna and Kocher 1995). gene, these workers found the mean percent
AT Singer and Hickey (2000) found a postitive corre- to be 82.2% with the content highest in groups Contributions to Zoology, 70 (I) - 2001 33
Number of transitions number of transversions in all of 16S rRNA, and 12S rRNA versus pairwise comparisons COl, sequences corrected for sequence-length variation(Pentcheff, unpublished). COI first, second, and third codon positions (COl 1,2,3) are summarized lust column (a-e). COI first and second codon positions (COI 1,2) are represented in the second column (f-j). 16S rRNA and RNA are summarized in i the third and fourth columns, k-o and p-t, respectively. This figure gives an indication of the extent of
si ton/transversion and bias the extent ofsaturation in substitutions among different hierarchical levels. Some points overlap one another. 34 R. Wetzer - mtDNA variation in isopods
considered be in the to relatively recently diverged sequence divergences equal or exceed those cal-
hymenopteran phylogeny, i.e., bees, chalcidoids, culated for suborder comparisons (see Figs. 1-3;
scelionids, and some endoparasitoid brachonids. COI amino acids, COI nucleotides, and 16S rRNA).
found that bias For the 12S rRNA the six cirolanid Additionally, they base-composition gene, sequence
As Ts the reflected the substitution bias toward and at comparisons were highest divergences mea-
different hierarchical levels. sured, with dissimilarity values ranging from
47-52% (Fig. 4). These comparisons represent
minimum and maximum divergence values, and
Transition/transversion bias comparisons were used only once (i.e., taxa used
in a generic pairwise comparison were not reused
in The The transition-to-transversion ratio (ti/tv) is an suborder pairwise comparisons). lower di-
values important aspect of models of sequence evolution vergence seen for Asellota, Oniscidea,
because it the and Serolidae expresses relative probabilities of Cymothoidae, are most probably an
different types of nucleotide changes based on their artifact of the few species available for this study.
In structural class. Nucleotide substitution within a contrast, the most speciose suborders of iso-
also have the structural class (transitions) occur with greater fre- pods greatest sequence divergences.
quency than changes between structural classes Nearly 20% of all described isopod species are
(transversions; Li and Graur 1991). Wakeley (1996) Flabelliferawith the Sphaeromatidae representing
summarized current models used to correct for 6.4% and Cirolanidae 4.2% of all species. Phrea-
genetic distance and argued that the knowledge of toicids and valviferans represent only 0.9% and
transition bias facilitates inferences about mutational 5.6% of named isopods, respectively.
of patterns and about the type and strength natural Sequence divergence for mitochondrial genes
selection. Although transitions occur more fre- among crustaceans is highly variable. Among
quently than transversions, with the passing oftime, infraorders of clawed lobsters (Astacidea) and
hermit crabs for multiple changes occurring at the same nucleotide (Anomura) a 350-bp fragment of
position make it impossible to differentiate between the 16S rRNA gene, Tam and Kornfield (1998)
and found little 26.1% single multiple changes, i.e., saturation. This as as sequence divergence,
phenomenon obscures the “signal” sought in phylo- whereas Mannerand Fugate (1997) observed 46.8%
reconstruction for genetic (Hillis 1991). Purvis and sequence divergence a 275-bp fragment of the
Bromham (1997) correctly point out that although 12S rRNA gene among infraorders of anostracans
the choice of the tree can be affected by the value (Branchiopoda). of ti/tv, this value is rarely specified in published
molecular phylogenies.
Rate variation across lineages
mutation Sequence divergences Although rate may vary among nucleotide
sites within a gene, Yang (1996) attributes the major The taxa surveyed in this study revealed a consis- reason for this variation to different selective con- tent pattern in which specific lineages always had straints at different sites owing to the functional
whereas other and/or structural of the relatively large sequence divergences, requirements gene or protein.
lineages had consistently smaller divergences (Figs. Despite the attractiveness of a standardized temporal
scheme of 1-4). These are similar for all three classification for extant patterns genes. biological spe-
In the smallest cies and Johns general, sequence divergences were (Avise 1999), such schemes do not observed for membersofthe suborders for Phreatoici- account highly unequal evolutionary rates among dea and Valvifera, For and the largest divergences lineages. example, Britten (1986) measured a observed among members of the flabelliferan fami- 5-fold rate change between different vertebrateand lies and Cirolanidae. For invertebrate Sphaeromatidae example, groups. Rodents, sea urchins, and in some generic comparisons of Cirolanidae the Drosophila have the fastest evolving DNA, whereas Contributions to Zoology, 70 (1) - 2001 35
higher and some bird have the from COI be desirable primates lineages positions sequences may
slowest. These differences in rates have been for studies at- phylogenetic at the genus, family, and tributed variation in biochemical to mechanisms suborder level, and using COI amino acids in family-
such DNA as and DNA that can and order-level studies replication repair phylogenetic may also be be differentially active taxa (Britten a useful The 16S rRNA be among 1986). strategy. gene may most Cacconeand Powell (1990) calculated absolute rates appropriate for studies addressing populations, of in change DNA as ca. 5-10 times and ofValvifera Drosophila species, genera and Phreatoicidea,
faster than what is found in and and most vertebrates, possibly closely related genera ofFlabellifera. this holds for the more conservative of the 12S rRNA data part Although the set was the smallest
nuclear that genome. They point out morphological of the three, this gene may best be restricted to
similarity, chromosomal similarity, and/or ability population and species level studies within to form interspecific hybrids is often associated Phreatoicidea and Valvifera and used cautiously with quite high levels of single-copy DNA diver- for others.
gencein insects as to mammals and birds. The information compared phylogenetic content of gene
Bermingham and Lessios 23 be (1993) using protein sequences can improved by various sequence
loci and restriction endonuclease analysis, found alignment strategies such as exclusion of alignment-
that sea urchins Diadema ( ) across the Isthmus of ambiguous nucleotide sites (Gatesy et al. 1993) or
Panama its (~3 mya since origin) were evolving at by successive weighting strategies of alignment-
a 10-fold order of slower than those of the “elision” magnitude ambiguous sites, e.g., by method
the two urchin genera Echinometra and Eucidaris. (Wheeler et al. 1995). Phylogenetic noise can also
The of the urchin elegance study derives from the be reduced by assigning different weights to certain known divergence times and the ability of the classes of nucleotidesubstitutions (e.g., transitions,
authors to rule out and sampling error, mass mortality transversions, compensatory substitutions) into and well and subsequent population “bottleneck”, as parsimony maximum-likelihood analyses
as differences in In generation times contributing to (Swofford et al. 1996). the present study, in which this In evolutionary rate change. an example from the phylogenetic relationships are unknown,
crustaceans, Manner and Fugate’s (1997) I2S rRNA discerning between loss of phylogenetic signal
branchiopod found the least of from study amount se- resulting sequence divergence and loss of phy- quence of all intraordinal result of divergence comparisons logenetic signal as a inappropriate taxo- to be between the notostracan genera Triops and nomic rank is not possible. Combining multiple Lepidurus considered (both “living fossils” andboth congruent data partitions would not only have the unchanged from the effect of the morphologically Triassic, 225 increasing size of data sets and thereby m ya). In the cladoceran but contrast, genera Daphnia phylogenetic signal, techniques such as six- and Moina were found to have sequence divergences parameter parsimony attribute a cost to each type twice those of and have of transformation based notostracans, they recog- on its observed frequency nizable fossils known from the Miocene (24 mya). in each separate data partition. The latter method
shown has been to consistently improve the positive
between and relationship congruence accuracy of Conclusions known phylogenetic relationships (Cunningham
1997). he most In speciose isopod suborders, wrought with designing an order or suborder level molecular the highest levels of can be the bane for a unstudied homoplasy, phylogenetic study previously group
°f a morphological existence. within the systematises On a Crustacea, my recommendations would molecular level, these also have include: collection of a minimum same groups may (I) two to four tic most be divergent mitochondrial nucleotide se- species or genera thought to most divergent, quences. For all three members of the sub- i.e., related include genes, most distantly (this may taxa oider Flabellifera have the most divergent DNA. which are the most speciose, have unusual lifestyles, used on the ti/tv ratios (Fig. 5), eliminating third morphology, and/or have had a tumultuous taxo- 36 R. Wetzer - mtDNA variation in isopods
S, Bankicr AT, Barrel! BG al. (14 coauthors). nomic history); (2) obtain as equal a representation Anderson ct 1981. and of the human mitochon- of (he Sequence organization taxa across group as possible; (3) survey drial Nature 290; 457-465. genome. two to three genes, if possible, (alignment problems Avise ,IC, Johns GC. 1999. Proposal for a standardized are greatly reduced by using protein-coding genes; temporal scheme of biological classification for extant nuclear out single copy genes); (4) carry preliminary species. Proc. Nat. Acad. Sci. 96: 7358-7363. alignments, check data for nucleotide bias, ti/tv Bcrmingham E, Lessios HA. 1993. Rate variation of protein
and mtDNA evolution as revealed by sea urchins separated ratios, and saturation levels before committing to by the Isthmus of Panama. Proc. Nat. Acad. Sci. 90: 2734- a large-scale sequencing effort. 2738.
Blouin MS, Yowell CA, Courtney Cl I, Dame JB. 1998.
Substitution bias, rapid saturation, and the use of mtDNA
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