<ooloszcal Journal of the Linnean Sock& (1996), 116: 6 1-69.

With 2 figures

Tardigrade biology. Edited by S. J. McInnes and D. B. Norman

@

Downloaded from https://academic.oup.com/zoolinnean/article-abstract/116/1-2/61/2684288 by guest on 18 November 2018

Phylogenetic position of the Tardigrada based
on the 18s ribosomal RNA gene sequences

SEUNG Y E 0 MOON AND WON KIM

Department of Molecular Biology, Seoul National UniversiQ, Seoul 151-742, Korea

The phylogenetic position of the Tardigrada remains uncertain. This is due to the limited information
available, and the uncertainty ofwhether some characters are homologous or analogous with other taxa.
Based on some morphological characters, current discussion centres on whether the taxon branches
from the annelid-arthropod lineage, or lies within the arthropod complex. The molecular data presented
here from an analysis of the 18s rRNA gene sequences are used to test the validity of these two
hypotheses. Phylogenetic inference by the maximum parsimony and distance (neighbour-joining)
methods suggests that the Tardigrada is a sister group of the major protostome eucoelomate assemblage
that emerged before the arthropods, annelids, molluscs, and sipunculids evolved. The tardigrade clade
also appears as an independent lineage separate from the nematode clade, thus supporting the current
idea that tardigrades do not have a close aschelminth relationship. The molecular data also imply that
several morphological features, considered significant in determining the phylogenetic relationships of
tardigrades, are not synapomorphic characters.
O199F The Iinnean Society of London

ADDITIONAL KEY WORDS: -molecular phylogeny - 18s rDNA.

CONTENTS

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Material and methods . . . . . . . . . . . . . . . . . . . . . . . 62
Taxa compared . . . . . . . . . . . . . . . . . . . . . . . 62
DNA extraction, PCR amplification, cloning and sequencing . . . . . . . . 62
Phylogenetic analysis . . . . . . . . . . . . . . . . . . . . . . 63
Results and discussion . . . . . . . . . . . . . . . . . . . . . . . 63
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . 65
References . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

INTRODUCTION

Microsopic size (50 pm-1200 pm) and a small number of distinctive morpho-
logical characters have led to relatively few taxonomic works on tardigrades (for
recent reviews, see Schuster et al., 1980; Pilato, 1969, 1975, 1982; Bertolani, 1981,
1990, 1992; Kristensen, 1987; Nelson & Higgins, 1990; Nelson, 1982, 1991).
Although they were recognized as a phylum by Ramazzotti (1 962), their taxonomic
rank and phylogenetic affinity remain unresolved. They are considered as enigmatic
because some of their somatic characters are found also in other taxa, such as the
61
0024-4082/96/010061+09 $18.00/0 01996 The Linnean Society of London
Nematoda and the protostome coelomates (Annelida, Onychophora, and
Arthropoda). Moreover, the absence of clear fossil records and detailed embryo-
logical data for tardigrades has increased the difficulty of deciding whether these
characters are homologous or analogous with other taxa. Current discussion has
focused on tardigrades either stemming from the annelid-arthropod line or being
located with the arthropod complex, with a particular affinity to the Onychophora

Downloaded from https://academic.oup.com/zoolinnean/article-abstract/116/1-2/61/2684288 by guest on 18 November 2018

(see Marcus, 1936; Greyen, 1982; Hickman, Roberts & Hickman, 1984; Brusca &
Brusca. 1990; Kozloff, 1990; hleglitsch & Schram, 1991; Ballard et al., 1992;
Kinchin, 1992; Fortey & Thomas, 1993). However, the molecular information
presented here from an analysis of the 18s rRNA gene sequences offers a different
perspective on the phylogenetic position of the Tardigrada.

.\L.\TEKLU .UXD METHODS

Taxa compared

?'he taxa compared in the present study were tardigrade (Hypsibius sp.), nematode
(Caenorhabditis elegans), arthropod (Eurypelma cal$rnica, Artmia salina, Tmebrio molitur),
annelid (Chaptopkms sp.), mollusc (Gyptochiton stellen), sipunculid (GuYingia gouldiz) and
nemertine (Cerebratulus lactms). The platyhelminthes Lhgesia tiF'na was designated as
the out\group. The culture of live tardigrade Hypsibius sp. was obtained from Ward's
Natural Science International Marketing Group. The complete sequence of the 18s
rRNA gene of the tardigrade was determined in thc present study, and compared
with published gene sequences of the other nine taxa (Ellis, Sulston & Coulson, 1986;
Field et al., 1988; Hendricks et al., 1988; Turbeville, Field & Raff, 1992).

Du,Wextraction, PCR ampljfication, cloning and sequencing

Live individuals were washed with distilled water to remove all the external debris
and then starved to remove gut contents. The individuals were disintegrated in a
microtube using a sonicator for a few minutes, with intervals of 30 seconds for
cooling the tube on ice. The isolation of genomic DNA from the individuals was
performed using the method modified from Blin and Stafford (1976).The 18s rDNA
was amplified using PCR (Saiki et al., 1985, 1988) with two primers located at either
end of the molecule (5'-CCTGGTTGATCCTGCCAG-3', 5'-TAAT-
GATCCTTCCGCAGGTTA-3'). The thermal cycle parameters were 94OC, 1 min
(initial denaturation, 5 min)/52'C, 2 min/72OC, 3 min (final extension, 10 min). The
reaction was cycled 30 times. The amplified double-stranded 18s rDNAs were
extracted with equal volumes of phenol:chloroform, followed by purification with
GENE CLEAN I1 kit (BIO 101), and diluted in distilled water. For blunt-ended
ligation, both ends of the PCR products were modified using T4 kinase and T4
polymerase. The blunt ended 18s rDNAs were purified with the GENE CLEAN I1
kit, inserted into pUC 19 plasmid vector and transformed to DH5-a cell lines. The
double-stranded recombinant plasmid DNA was purified by PEG precipitation or
QIAGEN plasmid kit (Pharmacia). The DNA sequencing was performed by the
dicleoxy-termination method (Sanger et al., 1977) using Taq-Track kit (Promega),
with two vector primers pM13 universal primer (40),M 13 reverse primer (-2411 and
 PHYLOGENETIC POSITION OF TARDIGRADA 63

an additional fourteen primers as used by Spears and colleagues (1992). Sequencing

reaction mixtures were electrophoresed on buffer-gradient 6% polyacrylamide
gels.

Phylagenetic anabszi

Downloaded from https://academic.oup.com/zoolinnean/article-abstract/116/1-2/61/2684288 by guest on 18 November 2018

Sequence alignment (Appendix) was performed using the CLUSTAL V multiple
alignment program (Higgins, Bleasby & Fuchs, 1992) beginning at universally
conserved regions, and then checked by eye. T o avoid possible systematic errors,
regions of ambiguous alignment including positions exhibiting high length variability
were excluded from the phylogenetic analyses. Both maximum parsimony and
distance (neighbour-joining)methods were used for the analysis. The bootstrapping
method of data resampling (Felsenstein, 1985) was applied to evaluate the
significance of the result. For maximum parsimony analysis, the BRANCH AND
BOUND search option of PAUP, version 3.0s (Swofford, 1990)was used except for
bootstrapping. The HEURISTIC search was employed for the bootstrap test by
using TBR branch-swapping and CLOSEST stepwise addition. Ten trees were held
at each step. Alignment gaps and unknown bases were considered as missing data.
Neighbour-joining analysis was applied using PHYLIP, version 3 . 5 ~(Felsenstein,
1993). Evolutionary distances were calculated by the DNADIST program, using
Kimura two-parameter model with no transition/transversion bias. Bootstrapping
was performed with the SEQBOOT program, with a random input order of
taxa.

RESULTS AND DISCUSSION

Maximum parsimony analysis generated a single minimum-length tree of 666

steps with 180 phylogenetically informative sites (Fig. 1A). The overall consistency
index is 0.748, with a consistency index excluding uninformative characters of 0.609.
The tree topology is largely concordant with the previous results based on the 18s
rRNA gene sequence data in assemblages of the protostomes, including the position
of the nemertine Cerebratulus lacteus (see Turbeville et al., 1991, 1992).The tardigrade
Hypsibius sp. appears as a sister group of the protostome assemblage which includes
representative species of arthropods, annelids, molluscs, sipunculids and nemertines.
The tardigrade clade also represents a discrete lineage apart from the nematode
clade (Fig. 1A). This proposal is supported by bootstrap analysis (Fig. 1B).
Measurement of the reliability of the maximum parsimony phylogeny, by
considering all trees within 1% of the length of the shortest tree, also supports the
significance of the separation of the tardigrade from the major protostome
assemblage (Fig. 1C). As the nematode branch is faster evolving, and the maximum
parsimony analysis may generate an erroneous tree(s) when the rates of evolution
vary with the evolutionary lineage (Felsenstein, 1978; Nei, 199l), this parsimonious
solution was compared with that of the distance (neighbour-joining)method, which
is more efficient when the rate of nucleotide substitution varies with lineages (Nei,
1991; Olsen & Woese, 1993). The neighbour-joining analysis, with bootstrapping,
was also consistent with that of the maximum parsimony analysis, except for a little
variation in the placement of the sipunculid GoJingia gouldii and the mollusc
ti* S. Y E 0 XIOON rZND 1%’. KIhl

Gyptochiton ~tellcriwithin the protostome assemblage (Fig. 2). This variation may be
explained by the rapid diversification of members in the protostomes, and shows that
these 18s rRNA gene sequences are not useful for determining the positions of the
sipunculid and the mollusc (Turbeville et al., 1991, 1992).
The molecular data presented here propose that tardigrades originated before the
advent of the protostomcs, and dismisses the hypothesis that they have affinities with

Downloaded from https://academic.oup.com/zoolinnean/article-abstract/116/1-2/61/2684288 by guest on 18 November 2018

both or either of the annelids and arthropods. These data also imply that several
morphological features such as cuticle, muscle attachment, cephalic appendages,
and/or excretory osmoregulatory system (see Baccetti & Rosati, 1971; Crowe et al.,
1971; Bussers & Jeuniaux, 1973; Shaw 1974;, Greven & Groht, 1975; Wdz, 1979;
Kristensen, 1978; Dewel & Dewel, 1979; Greven, 1979; Kristensen, 1981; Greven &
Peters, 1986) between tardigrades and arthropods are not synapomorphies, but are
either plesiomorphies or results of convergent evolution (Fig. lA, B; Fig. 2).

A
6--- .4rtemia

1
4- Chaetopterus - a m e l id
arthropods

2- Cerebrat ulus - nemer t i ne

9- Cryptochiton - mollusk
-1 Golfingia - sipunculid
-4 Hypsibius - tardigrade
Caenorhabditis - nematode
0-4- hrgesia -flatworm

B
Artemia
C

Hypsibius
Caenorhabditis
Dugesia

Figure 1. Phylogenetic relationships generated from maximum parsimony analysis using PAUP. A,
Minimum-length phylogenetic tree. Numbers indicate the branch lengths at each node. B, Bootstrap
.in90 majority-rule consensus tree. Numbers at nodes represent the bootstrap percentages from 1000
samples. C:, 30?h majority-rule consensus tree of 101 trees lying within 1% of the length of the shortest
tree. Numbers at no& represent the frequency with which clades descending from nodes were found
among the 101 trees saved.
 PHYLOGENETIC POSITION OF TARDIGUDA 65

Tenebrio
Euryplma
Chaetopterus
Cerebratulus

Downloaded from https://academic.oup.com/zoolinnean/article-abstract/116/1-2/61/2684288 by guest on 18 November 2018

Golfingia
83
I Cryptochiton

Dugesia
0.05 0.1 0.15 0.2

Figure 2. Phylogenetic relationship generated from neighbour-joining analysis. Numbers at nodes

represent the bootstrap percentages from 1000 samples. Average number of substitutions per sequence
position is indicated by numbers at the top of the scale bar.

Tardigrades are coelomic animals (Marcus, 1929), and more detailed study on the
origin of the mesoderm could provide a clue for finding the synapomorphic
character(s) shared by tardigrades and the other major protostomes.
The tardigrade clade is also separated from the nematode clade, which supports
the current idea that morphological and physiological similarities (e.g. cellular eutely
of epidermis, structure of anterior foregut, pharynx and oesophagus, reduction of
coelom, absence of circulatory and gas exchange organs, and/or cryptobiosis)
between tardigrades and some aschelminthes have been acquired due to their similar
life styles in similar habitats (Marcus, 1929; Brusca & Brusca, 1990). However,
whether tardigrades simply retained the aschelminth characteristics (although this
seems more parsimonious given the trees shown in Figs 1 and 2) or acquired them
through a separate evolutionary track remains uncertain. Future comparative
analysis of the 18s rRNA gene and other molecules could provide additional
evidence for resolving the origins of these characters. Despite the molecular data
from this study and the few known morphological characters, the data require
further morphological and in particular embryological support.

ACKNOWLEDGEMENTS

We thank Drs G. S. Min for technical assistance in sequencing the 18s rDNA, C.
B. Kim for discussions, and S. R. Gelder for providing helpful suggestions that
improved the manuscript. This work was supported by grants from KOSEF in
1991-1994, SRC (94-4-2) and the Ministry of Education of Korea (Institute for
Molecular Biology and Genetics) in 1994.

REFERENCES

Baccetti B, Rosati F. 1971. Electron microscopy on tardigrades. 111. The integument. Journal of Ultra.stmcture
Research 34: 214-243.
6G s. Y E 0 hfOON AND w.KIM
BallardJW, Olsen GJ, Faith DP, Odgers WA, Rowell DM, Atkinson PW. 1992. Evidenc:e from 12s
ribosomal KXA sequences that onychophorans are modified arthropods. SrMre 258: 1345-1 348.
Bertolani R. 1981. The taxonomic position of some cutardigradcs. BoflPl/ino di ~ o o l o & 48: 197-203.
Bertolani R. 1990. 'l'ardigrada. 111: Adiyodi KG. Adiyodi RG, cds. Keprodudzz!e Iliolog ?/ In71ertebr&s, Il', U .
Chichrster: John iViley, 49-60,
Bertolani R. 1992. 'l'ardigrada. In: Adiyodi KG. .4diyodi RG. eds. Reproductive Biology ofInvertehrate.s, 1.' Chichester:
John Wiley, 255 266.
B h N, Stafford DW. 1976. .\ general method for isolation of high molecular weight DNA from eukaryotes.

Downloaded from https://academic.oup.com/zoolinnean/article-abstract/116/1-2/61/2684288 by guest on 18 November 2018

.\IUclek ilcids Research 3: 2303.
Brusca RC, Brusca GJ. 1990. Iniwttbrales. Sunderland, hM: Sinauer Associates.
Bussers JC, Jeuniaux C. 1973. Chitinous cuticle and s)-stematicposition of Tardigrada. Biochmiral Syste7natirs 1:
77 78.
Crowe JH, Newell IM, Thomas WW. 1971. Finc structure and chemical composition of the cuticlc of the
t a r d i p d c , .\larrohio/zu. orprrlah Murray. Jounial de .\firrosc.opie 11: 107-1 20.
Dewel RA, Dewel WC. 1979. Studies on the tardi!grades. IV, Fine structure of hindgut of A/ilne.sium tnrdigradrrm
Doyire. Jortniai qf.\fotpholug 161: 79-1 10.
Ellis LEYSulstonp, Coulson AR. 1986. The rDN.4 of C. elegam: sequencr and structure. .Nurki.Acid Rerearrh
14: 2:3+5--2361.
Felsenstein J. 1978. Caw. in which parsimony or compatibility methods will be positively misleadinq S~~~stematzr
.;)ofop?. 27: 401 ~410.
Felsenstein J. 1985. Confidence limits on the phylogenies: an approach using the bootstrap. Eriulu/ion 39:
i8:3--791.
FelsensteinJ. 1993. PHYLIP (Phylogeny Inference Packagej. Version 3 . 5 ~Department . of Grnetics, University
0 1 IGsshirigton. Seattle.
Field KC, Olsen GJ, Lane DJ, Giovannoni SJ, Ghiselen MT, Raff EC, Pace NR, Raff RA. 1988.
3lolccular phylogeny of the animal kingdom. Srienrr 239: 748- 753.
Forty RA, Thomas RH. 1993. The case of the velvet worm. Aafure 361: 205-206.
Greven H. 1979. Notes on the struciure of vasa h.lalpighii in the eutardigradr I s o ~ J ~ .augvsti s ~ ~ ~(Murray,
u . Y 1907).
In: \Vrglarsl;a B? cd. .Second intrmationol gmpositrrn on hrdipades, hiakour, Poolond, 3ub 28-30, 1977. <es<y&Naukowe
l;lialer?~.tptu Jagiellorisliego. Bare <oolo&zne 25: 87-95.
Greven H. 1982. Homologous or analogous? A suney of some structural patterns in the Tardigada. In: Nelson
DR, ed. tfo(-eedings of'thr third irrtrmat;onal pniposiuni on /he Tardkada. dugus1 3-6, 1.980. 30hnron CiQ, Tennessee, UX4.
East Tennessee State Yniversity Prcss, 55-76.
Greven H, Peters W. 1986. 1,ocalization of chitin in the cuticle of Tardigrada using wheat germ agglutinin gold
conjugate as a sperific elrctron dense marker. fi.rsur and Cell 18: 297--304.
Greven H, Groh6 G. 1975. Die Feinstruktur des Intepmentes und der Muskelansatzstellen von Echiniscoicles
. i p ~ u t i d i(Heterotardi<grada).He&olander I.l~.ssmrhafiliched/eeremntermchungm 27: 450 460.
Headricks L, van Broekhoven C, Vandenberghe A, van de Peer Y, de Wachter R. 1988. Primary and
sccondar). structure of the 18s ribosomal R i A of the bud spider E q p e l m a ral$micn and evolutionary
relationships among euk;n-)-otirphyla. European 3ounial of Biorhmisly) 177: 1 5 20.
Hiclanan CP Jr, Roberts LS, Hickman FM. 1984. Ititmgruted p r i n r i f h oJ;oo/ogy. hfosby College Publishing.
Higgins DG, Bleasby AJ, Fuchs R. 1992. Clustal V-improved Fofiware for multiple alignment. CABIOS 8:
189---
191.
Kincbin IM. 1992. iVhat is a tardigrade? 2\/zrro.rco/y 36: 628 634.
Kozloff EN. 1990. Incwtebrnfts. New York Saunders College Puhlishing.
Kristensen RM. 1978. On the structure of Uutif1zpe.rnoveziaq' Kristensen 1976.2. The muscle-attachments and the
true cross-striatcd rrruscles, :uol+cho .4nz:piger 200: 173-184.
Kristensen RM. 1981. Srnsc o r p i s of two marine .\rthrotardigradcs (Hctcrotardigrada, 'l'ardiqada). dcta
<oolugii~a 62: 27 4 1 .
Kristensen RM. 1987. Generic rrsision of the Echiniscidae (Heterotardigrada), with a discussion of thc origin of
<.
the Sandy. la: Brrtnlani R. rd. Biolop . f / h e fnrd&aiips. Stlecfcd Fmposin and nionopphs U, I., 1. Modena, Italy:
Murchi, 26 Ib335.
Marcus E. 1929. Tardigrada. In: Rronn HG, ed. hlassm und Ordnungm des Em&ch, 4. Akademische
\'rrlagsgeseUsrhaft, Ixipzig, 1 608.
Marcus E. 1936. Tardigrada. In: Schultze F, cd. Das T m c i r h . \raker de Gruyter, Berlin.
Meglitsch PA, Schram FR. 1991. Inrpllpbrate : o o l q ~Oxford: Oxford University Press.
Nei M. 1991. Rrlatii,e etficiencies of different tree-making methods for molecular data. 111: Miyamoto MM,
Ciacraft rds. Pl~slo~gme/ir
J? ana!yric ofD.\A requoirer. Sew York Oxford University Press, 90 128.
Nelson DR. 1982. Dcvclopmental biology of the Tardigrada. In: Harrison Fw,Cowden RR, eds. ZIPr~elopnzentul
h r o l g . qffierhwoler t n w r k b r a k Sew York: .Van R I.iss. 363 ~368.
Nelson DR. 1991. Tardigrada. In: Thorp JH, Cosich AP, eds. Erolugy and rlasx$cation OfNorth Amm'can jieshuiuter
i r m r / ~ 6 r 0 t zLondon:
~ Acadrmic Press, 30 I 52I .
Nelson DR, H i e s RP. 1990. 'I'ardigrada. In: Dindal D, ed. Soii biology guide. New Yorl: Wiley, 393--419.
Olsen GJ, Woese CE. 1993. Ribosomal KWA: a key to phylogeny. 77uFrlSEBjounzal, 7: 113- 123.
Pilato G. 1969. EvoluLione e numa sistmiaziorie drgli Eutardigrada. Hollettino di <oologia. 36 3: 327-345.
 PHYLOGENETIC POSITION OF TARDIGRADA 67

Pilato G. 1975. On the taxonomic criteria of the Eutardigrada. First international symposium on Tardigrada,
Pallanza, Italy, June 17-19, 1974. Mmorie dell’Istitut0 Ituliano di Idrobwlogza 32: Suppl.: 277-303.
Pilato G. 1982. The systematics of Eutardigrada. A comment. xeit.schijt$r zoologkche Systaatik und Evolutwnsforschun~
20, 4: 271-284.
Ramazzotti G. 1962. I1 phylum Tardigrada. Memorie dell’ Istituto Ituliano di Idrobiologia 16: 1-595.
S a i k i R, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA. 1988. Primer-
directed enzyme amplification of DNA with a thermostable DNA polymerase. Science 239: 487-489.
Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N. 1985. Enzymatic

Downloaded from https://academic.oup.com/zoolinnean/article-abstract/116/1-2/61/2684288 by guest on 18 November 2018

amplification of P-globin genomic sequences and restriction site analysis for diagnosis of sic!& cell anemia. Science
230: 1 3 5 w 354.
Sanger F, Nickeln S, Coulson AR. 1977. DNA sequencing with chain terminating inhibitors. Proceedings ofthe
Ndional Academy 0fSc;mCe (USA) 74: 5463-5467.
Schuster RO, Nelson DR, Grigarick AA, Christenberry D. 1980. Systematic criteria of the Eutardigrada.
Transactions of the American Microscopical SocieQ 99: 284-303.
Shaw K. 1974. The fine structure of muscle cells and their attachments in the Tardigrade Macrobiohu hufelandi.
Tissue and Cell 6: 431445.
Spears T, Abele LG, Kim W. 1992. The monophyly of brachyuran crabs: a phylogenetic study based on 18s
rRNA. Systematic Biology 41: 446-46 1.
Swofford DL. 1990. PAUP Phylogenetic Analysis Using Parsimony, Version 3.0s, Illinois Natural History Survey,
Champagne.
Turbeville JM, Heifer DM, Field KG, RalTRA. 1991. The phylogenetic status of arthropods, as inferred from
18s rFWA sequences. Molecular Biology and Evolution 8: 66%686.
Turbeville JM, Field KG, RalTRA. 1992. Phylogenetic position of phylum Nemertini, inferred from 18s rFWA
sequences: molecular data as a test of morphological character homology. Molecular Bzolog~ and Evotutzon 9:
235-249.
Walz B. 1979. Cephalic sense organs of Tardigrada. Current results and problems. In: Weglarska B, ed. Serond
intaational gmposium on turdkades, Krakow, Poland, 3 u Q 28-30, 1977. Zeszyty NaNaukowe Uniwergtetu Jagdoriskiego,
Race <oolog&ne 25: 161-168.
 * 07 *50 *300
Artemia salina . . .dATTAGATCA4GACCA4. . .
. . .T G G T C h T G A A T A A . . . . . . G C A C G X T C T C G C A C C C G C C C ' X C T C W T C ~ A A T G T C T C A
Euryplma californica . . TGGTGACI'CTGTATAA. . . G C A C G C C l T X G T C C C G C G A C G C A T C W T C ~ ' X C T C T ~ C C l T A T C A A C T G T C G A T G ~ A G ~ A T ~ ~
. . .TTATTAGACCAAAACCAA. . , . . ..
TTATTAAAACAAAACC A A . . .
. .. TGGTGACTCTCAATAA ... ...G C A C G ~ C T C G T A C C G C G C A G A T C W T C ~ G T C T C T G A ~ A T C A G ~ X X ~ G A G A G ~ A X ~ T ~
%%&d%s elegans . . TTATTAGMCAXCACCAA. . ,
. . . TGTTGACTCTGAATAA. . . . . G T X C A G X T T T C G X A C T G A C T C T A T X C C G G A A A G G T C T C T G X C C C ~ C A A C T A ~ G A T ~ A ~ A T T ~ G A C T
Tenbrio rolitor
.....
. .TTATTACATCAAAACCAA. . ,
. . .TCGTGACI'CTGAATAA. . . . G C A C G X T C T T G C A C C U j C G A C C C A T C m C X A A T G T ~ ~ C ~ T A T C A A ~ ~ ~ A T ~ A ~ X C T G C G ~ T
.
Chaetopterus sp . , .TGGTCACTCTGGATAA. .. . . , C C A T G X C C T C G A C C C C G C G A C C T A T ~ C ~ A A T G T C T G X C C C T A T C A A ~ C G A T ~ A C ~ G A T A T ~
. . .TTATTAGATC.MAACCAA. . .
Crypfochiton st'e1Ier-i . , .TGGTCATTCTNAATAA. . . . , CCACCCGCCTCCCCCCCGCGACGTATCWTC~ACTCTCT~CCCTATCAA CGATGGTACGXTGCIAT
. . .TTATTAGATCAAGATCAA. .. .
Golfiwiapuldii , . . TTA~AGATCMACCAA. ..
. . TK~TGAcTCAGAATAA. . . . . . C C A T C G X C C C A G A C C T ~ C C A ~ ~ ~ ~ ~ T ~ C G ~ ~ ~ C T A T C A A ~ ~ A T = ~ G ~ C G C A A T
.
esia tigrina . . .TGATCACTCTGGATAA.
, . .TTATTAGATCAAAATCAA, , . . . . CTACGACCI"IAGTGTTGACCACATATCTClTGAACTGCCT~CCCTATCAA~CGATG~AAGAT~A~
Eebratul us lacteus . . . TGACGAATCTNCATM. . . . GCACCCCCCTCGCCCCGCGCCTATCTTTC~AATGTCN~CCCTATCA4A~CCTAGGCCG~GACAT
. . .TTATTAGCTCAAAACCAA. , . :
*350 *400
Artemia salina ATCGTTGCAACGCCTAACGGGAATCGGffiTTCGATTCCGGAGAGGGAGCCTGAGAAACGG~A
Eurypelma caf jfornica ATCCTCXTAACGGGTAACGffiGAATCAG~CGATT~GGAGAGGCACCCTGAGAAACGG~A
H ibius sp AACGCGCTTACCCGTGACGGGAATCAGGTCCGATT~GAGAGGGAGCCTGAGAGACGG~A
d%chabdi <is el egans ATGGTTGTTACGGGTAACGGAGAATAAG~TCGA~CCGGAGAGGGAGC~AGAAA~GCTA
Tenbrio moll tor ATCGTTCTAACGCCTAACGGGAATCAGGGTTCGATT~GGAGAGGGAGCCTGAGAAACGGCI'A
Chaetopterus s p ATGGTTCTAACCG'XAACCGGAATCAGCGTTCGATTCCGGAGAGGGAGCATGAGAAACGGCI'A
CryptochitM stelleri ATGCTTATAACCGCTAACCGAGAATCA~TTCGATTCCGGAGAGGGAGCATGAGAAACGGCI'A
Colfirgia puldi i TAGCTIT?TACGGCTCACGCGGAATCAG~TTCGATTCCGGAGAGGAGCATGAGAAACGGCTA
Dugesia tigrina ATGGTTCTAACGGCTAACGGG~TCAGTGTTCGATTCCGGAGAG~AGCCTGAG~ACGG~A
Cerebratulus lacteus TNC~CTTACGGCTAACGGGAATCAG~CGA~CCGGAG~GGAGCATGAGAAACGG~N
*500
Artemia salina AATAACGATCCAGGACTCATCCGAGCCCCTCTCATlCGAATGA'XACACWTA4ATC~. , .
Eurypelma californica AATAACAATACGGGACTCGAGACCCCGTAATTGGAATGACTACACTCTAAATCC~.
H sibius sp AATAACGATGCGAGAGXCTACTAGClTTTCCTAATCGGAATGGGTACA~TAAATCCTTT. :
&nor$abdi {is elegans AATATAAAGACI'CXATCCTITTCCAXXTGAGI'TATITCAATGACrTGAATACAAATCATTC. ..
Tenbrio no11 tor AATAACGATACCGGACTCATCCGAGGCCCCCTAATCGGAATGA~ACACTCTAAA~m. .
Chaetopterus sp AATAACAATACGGGACTCTATCGAGCCCCCGTAATTGGAATGA'XACAA~AAATC~T. . ..
Cryptochit~ stelleri AATAACAATACGGGATCTCTN~G~CCCGTAA~GGAATGA'XACA~AAATCC~TT. ..
Go1fiGgi a p u ldii AATAACAATACCGGACTCTTACGAGCCCCCGTAATTGGAATGACTACAC~AAAT~TTT. ..
Llugesia tigrina AATAACAATATGCGXXXCCCACTGGTTTCATAATTGCAATGAGAACAT~~ATAC~T.. .
Cerebratulus lacteus AATAACAATACGGGACTCTATCGAGGCCCCG~ATTGGAATGA~NCA~AAA~CTTT. ..
*850 *900
4rtemia salina ATGCMTC?TGAATAGCAC~~GTCTT4TT.\TCTTCCT~\ACTGG4~XGAGCT4ATG~T~4CAGAG4CAG40(M;CGGC4TTCCTACTCCG?CGCfAGAGGTGXAAlTCTTGGACC~CGCAAG~
Eurypel ma cali fornica ~TGGMTAATCG1AT4ffiAC~TTCI'~TTTTCTT~CCGX4TACXGAG'XAATG4TT~GACGG4CAG4~G~G~4TTCCTATTGCGACCCTAGA~G~TT~GGACCCT~
H sibius sp ?TGC~TA1TGGA~T~W;ACCTCGCTICT4T~GTTffiTmCGG?GCTC~G4GGTAATGATT~4GAGG~CAG40(GffiGAC4TTC'XATGCGGCCTTAGA~GA4ATTCG4TC'XC~~G~
C"%orhabdi t i s ele5ans 1TGCAATAATGAXdC4GACCGGTIC~?TIT~GTTCT~C21~~CI'C?TTT4ATCCA1GAGGG.\C~~GGGGC4TTC'XATCATTACGCGAGAGCTGA~TTC~~ACC'X4~G4C
Tenbrio moli tor ?TGC4ATUTG.\dT4GACCTCG~CTAT~CTTCGTTTTCGG~4TTTTG4GGTA.\TGATTA4TAGG~CffiATGCGG~4TTCCT4TTGCGAM;~AGAG~GAAATT~GGATCCTC~A4G
Chaetopterus sp 4 T C G I U T X 4 T G G . \ . \ T A G A C C T C G G ~ C ~ ~ ~ ~ T G T ~ T C ~ ~ C T T X G A G ~ A ? T G A T T A A G~GAO(GffiGGCATTCGT4TTACGGTGI'TAG4GC;M;AA4TT~GGATC~C'XA4G
AGGGAC
Cryptochiton stelleri ~TCGUTA4Tffi4.\CCAGACCTCGGITCTAT~'XTGGTTTTCGG.UGTCXGAGCTA4TCATT~GA~GGAC~GA~GGGG~ATTC'XATTACGCT~AG4G~GA~TTCTTGGATCGCC'XA4G
Go1fingia gouldii 4TGCA?TA4TG44~TATAGG4CCTCC~CR\~TCTT~~TCTG.2ACCAXC.\GCTA4TC4TT~GAGGGACAGA~GUjGCCATICCT4TT4CCCTGTTAG4G~CAIUTTCGGATCCCCCTAAC
Dugesia tigrim ATGCMTlWTA~41TTffi?CTTM;~V\TATTTTGT~~CGA~C~G4AGTA4TGATT~4AG.\GACTGCCXGffiGCC4TATCTATGCTGCTGCTAGAGCTG~TT~4GATCATCACCAG
.Cerebratulus lacreus ATGGA4TMTCG4AT~GACCTCCGTTCTVTTTT\ITfGrm"lCCG~\ACTCXGAGG~ATGATTA4GAGGGACUj4CXCCGCGCA~CCTATTACG~CTTAGA~G~TTCTAGGATCGCC'XAAG

Sequenres of 18s r R N 4 genes used in the analysis. The position number is the nucleotide numbering of A. ~ u h u

Downloaded from https://academic.oup.com/zoolinnean/article-abstract/116/1-2/61/2684288 by guest on 18 November 2018

 APPENDIX (continued)

*y50
Artemia salina C C A A C 4 A C T G C C A 4 C A 4 C I T T C C C A A G ~ A T ~ T C A T T A A T C A A G A A C ~ 4 G T T A*loo0 GAT~A4CCAGCGAT~GCGGAC~~
G 4 M ; I T C C 4 A U ; C G A T C A G A T A C C G C C C T A ~ C T A A ~ A T ~ 4 C *lo50
E u r m l m a californica CGAACTACTCCCAAAGCATTGCCA4GAATCITTTCA~AATCAAGILZCG~GTAGAG~CG4A~GATCAGATACC~CT4~CT~~AT~4CGAT~CAACCAGCGAT~~GA~~
H slbius sp CCV\CTACTCCCAAACCATGTCA4G~TGCTTCCATTAATCAAGILZCGAA4G~AGAG~CG4AffiCGATCAGATACC~C~4~CTAA~AT~4CGATKCA4CC~GAT~~C~~
&norjmbclitis elegans CKCC~CAGCCAAACCATGCCA4G~TCT~CATTAATCAAGAACG~~CAGAGCITCG4A~GA~AG4TACCGCCCTA~~A~GTAAACGAT~CA~~CGA~GXG
Tenbrio mol I tor CffiACAGAACCGAAACCATCCCAAA4ACGCTTTCATTGATC4ACILZCG~A~AGAGCITCG4A~GATCAGATA~GCCCT4GTCTAA~ATAAACGATG~A~AGCGAT~G~GAC~l
Chaetopterus sp CGAAWA~GCCA4AGCATGCtAAG~T~CATTAATC~GAGCG~~CAGCG~CG4AG4CGATCAGATACCGTCGTA~CTGA~AT~CGATKCA4CTAGCGAT~G~~
Cry tochi ton stkl I eri CCAACTACTCCGA4ACCA~G~AAG~AT~TCATTAATCAAGAACG~AA~CAGAG~CG4AGACGATCAG4TACC~CCTA~CTGA~AT~CGAT~CA4CTAGCGAT~~~A~
GoPfiingia gouldii CGAACGACTGCCA4AGCATTG~AAG~4TGTTTTCATTAATCAAGAACG~A~CAGAGCITCGAAGACGATC~GATACC~C~AGTCTGA~ATAAACGAT~AA~AGCGATCGG~~ACITi
esia tigrina CA4ACNACTGCGAAAGCATTTG~AAGAATGTTTTCATTAATC~GAACGA~~CAGAGGATCG4AGACGATCAGATACC~CCTAGT~A~ATAAACTAT~AACTGACA~C~AA~~
Zebratulus Jacteus CffiACCCCTGCCA4AC~TTGCCAAGC4CGTTTTGATTACTCAAGAACGA~A~CAGAGCITCG4AGACGAT~GATACCG~~AGTTC~A~ATAAACGAT~GACTAGCGATCCG~~AGTTi
*I 100 CTGATGCC~AG4T~CCTG~C~CACGCCC~ACACT~AAG4A~AKG~
... A C C X C 4 T A T G A C A C T G A C C A 4 T A A C A G G T C T51450
rtemia salina CTTGAATCACTCCCCGCGCAGCTTCCGGGA44CCA...
urypelma californica CTCAAATCACTCCCCGGGCAGCTTCCGGGA44CCA... ...A G C X C C C 4 C G A G A C 4 C 4 G C A 4 T A A C A ~ T G A G A T G C C ~ A G A T ~ C ~ ~ C ~ C A C G C G C ~ A C A C T G A A G ~ T C A K G T G
sibius sp. 4TTCAATCACTCCACCGCCAGCTTCCGGGA44CCA. ... A C T X C C C 4 C G A G A T T C 4 G C A A T A A C A G C T C T C T G A T G C C ~ A G A T K C ~ G C C X G C A C G C C C ~ A C A C T G ~ G A A K 4 ~ ~
aenorhabdi cis elegans mMXXXKCCTCCCGAGCAGCTATCCGGA44CGA. ' ... AA GCC XCC XC AC4 CCG CA G4A CT TCC AAG CC AAT ATA 7C AC~ CAT GC TCC AGT GA CTC CAT TAA CG AAT ~GWGG ~TC ~CA TC G~~ ~G A AC ATC TG GCA ACG CG ~ T C A ~ ~ G
Tenbrio moll tor CTCCGATGACTCGCCCGCCAGCTTCCCCA9AtCA. .
: : ...
haetopterus sp. ClTCAATCACTCGKGCNCAGCTGCCGCCAAXCA... ... A C C X C A C A C C A G A W G A G C A A T A A C 4 G C T C T ~ G A T G C C C G
rpochiton stelleri TTNCAATCACTCCCCChhCAG~CCGGGAAACCA... ... ACCXCACACGAA4TCAGCATAACAG~CT~GATGCCCTTAGAT~CGGGCCXKACGCCC~ACAWGAAGGA~CA~~G
o fingia gouldii TTTNCITGACTCCKCGCCAGCTTCCCGGAAACCA ... ...A 4 C C C A T T C C A T 7 n ; C A C C A T A A C A G ~ C T ~ G A T G C C C T T A G A T ~ C C G G ~ C T G C A C G C G C ~ A C A A T G A A T ~ A T C A ~ ~ G
esia tigrina TTCAAATXXCTCXXXXXXX~CTTG~AG4ACTCA. . A~NAXATGAA4TGGACCAATATILZC4GCTCT~GAT~CCTAGATCTCCGGffiC~ACGCCC~ACAATGKA~A~A~GAG
terebra tulus lac teus CWCAATGACTCCCCCCNCNGCTTC~GG4AZCCA. . ... AGCXCACACG4GATTCAGCILZTATILZC~~T~GAT~CCTTAGAT~CGGffiCXGCACGCGC~ACACTGAAGGAATCAACGTGl
: ...

m
CD

Downloaded from https://academic.oup.com/zoolinnean/article-abstract/116/1-2/61/2684288 by guest on 18 November 2018