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Hydrobiologia (2006) 558:53–60  Springer 2006

J.R. Garey, D.R. Nelson & P.B. Nichols (eds), The Biology of Tardigrades
DOI 10.1007/s10750-005-1414-8

A family level analysis of tardigrade phylogeny

P. Brent Nichols1, Diane R. Nelson2 & James R. Garey1,*

Department of Biology, SCA 110, University of South Florida, 4202 East Fowler Ave, Tampa, FL 33620, USA
Department of Biological Sciences, East Tennessee State University, Box 70703, Johnson City, TN 37614, USA
(*Author for correspondence: E-mail:

Key words: tardigrada, systematics, cladistics, evolution, phylogeny, 18S rRNA

In the present study a character data set suitable for cladistic analysis at the family level was developed. A
data matrix consisting of 50 morphological characters from 15 families of tardigrades was analyzed by
maximum parsimony. Kinorhynchs, loriciferans, and gastrotrichs were used as outgroups. The results agree
with the currently accepted hypothesis that Eutardigrada and Heterotardigrada are distinct monophyletic
groups. Among the eutardigrades, Eohypsibiidae was found to be a sister group to Macrobioti-
dae+Hypsibiidae, while Milnesiidae was the basal eutardigrade family. The basal heterotardigrade family
was found to be Oreellidae. Echiniscoideans grouped with some traditional Arthrotardigrada (Renaud-
arctidae, Coronarctidae+Batillipedidae) suggesting that the arthrotardigrades are not monophyletic. The
18S rRNA gene sequence of Batillipes mirus Richters, 1909 and Calohypsibius schusteri Nelson &
McGlothlin, 1996 were obtained and their addition to a previously published dataset supports the
monophyly of Heterotardigrada and of Parachela versus Apochela within the Eutardigrada.

Introduction based on morphological characters that include:

claw size, shape, organization, and number; orga-
The phylogenetic position of Tardigrada has long nization of the bucco-pharyngeal apparatus; length
been debated (for reviews, see Ramazzotti & of stylets; size, shape, and number of placoids;
Maucci, 1983; Kinchin, 1994), and morphology has cuticular patterns and ornamentation; and
been used to assess the relationships among some morphology of eggs and spermatozoa.
genera within a few families (Renaud-Mornant, The tardigrade literature consists of a plethora
1982; Kristensen, 1987; Pollock, 1995; Bertolani & of species descriptions, ecological surveys, and
Biserov, 1996; Jorgensen, 2000; Guidetti & Berto- only a few limited systematic studies. An overall
lani, 2001). Tardigrades have previously been framework for tardigrade phylogeny will encour-
placed along an annelid-arthropod lineage, often age a wider application of morphological and
closely associated with the onychophoran–arthro- molecular studies to tardigrade evolution. For
pod complex, although there have been some example, a study of nematode phylogeny (Blaxter
arguments for placing them with the aschelminthes et al., 1998) was pivotal in encouraging new dis-
group (Dewel & Clark, 1973a, b; Kristensen, 1991). cussion and a reassessment of nematode system-
Current evidence suggests, however, that tardi- atics, particularly in light of the completion of the
grades, onychophorans, and arthropods form a Caenorhabditis elegans genome project.
monophyletic clade known as Panarthropoda Early molecular phylogenetic studies associated
(reviewed in Schmidt-Rhaesa et al., 1998). The tardigrades with arthropods (Garey et al., 1996;
current taxonomy of clades within Tardigrada is Giribet et al., 1996), and a leading hypothesis

places tardigrades, arthropods, priapulids, nema- relationships among the families within each tar-
todes, nematomorphs, kinorhynchs and loricifer- digrade order, to help determine if the families are
ans in a group of molting animals called truly monophyletic and to compare these analyses
Ecdysozoa (Aquinaldo et al., 1997; Mallatt & with results from a molecular dataset.
Winchell, 2002). Tardigrada, as a member of
Panarthropoda, will likely play an important role
in the future assessment and discussion of Materials and methods
arthropod evolution and development in the same
way that Onychophora has played a role in the Characters: A matrix consisting of 50 morpho-
understanding of protostome evolution (deRosa logical characters from current literature was
et al., 1999) and the evolution of body plans and scored for 15 tardigrade families (for review, see
appendages (e.g. Panganiban et al., 1997; Grenier Schuster et al., 1980; Binda & Kristensen, 1987;
& Carroll, 2000). Kristensen, 1987; Bertolani & Rebecchi, 1993;
Garey et al. (1999) found close agreement Schmidt-Rhaesa et al., 1998; Jorgensen, 2000,
between molecular and morphology based phy- Guidetti & Bertolani, 2001). Three outgroups, two
logenies that included five species of tardigrades, within Ecdysozoa (Loricifer & Kinorhyncha) and
suggesting that the characters for the current one outside Ecdysozoa (Gastrotricha) were
morphological taxonomy are appropriate. included in the analysis. Ten multi-state characters
Although tardigrade genera, families, orders, and were present. The character data matrix was
classes (Table 1) are each well defined by constructed using Nexus Data Editor (NDE)
morphological characters, the relationships among version 0.5.0 (
the families and among genera within the families rod/NDE/nde.html). Phylogenetic Analysis Using
have not been systematically evaluated. One Parsimony (PAUP*) version 4.6 (Swofford, 1998)
exception is the family Echiniscidae for which was utilized to find the most parsimonious trees.
several cladistic studies have been published Molecular: Deoxyribose nucleic acid was iso-
(Kristensen, 1987; Jorgensen, 2000). lated from individual Calohypsibius schusteri
The present study was designed to assemble Nelson & McGlothlin, 1996 and Batillipes mirus
and analyze potential morphological characters Richters, 1909 by mechanical maceration of the
that are informative at the family level, to test the cuticle in a 25 mg/ml proteinase K solution fol-
lowed by three freeze/thaw cycles of 20 min and a
Table 1. Higher level Taxonomy of the Tardigrada
denaturing step at 96 C for 15 min. Two primers,
Class Order Family GGTGAAACCTCGGAAGGATCA-3’ (Mallatt
et al., 2004), were used to amplify the 18S rRNA
Eutardigrada Parachela Calohypsibiidae
gene. Polymerase chain reaction (PCR) was
performed with denaturing at 95 C for 30 s,
annealing at 55 C for 20 s, and extension at 72 C
for 2 min for 44 cycles. The PCR product was
cloned using a commercial cloning kit, TOPA-TA
(Invitrogen) and sequenced using a CEQ 8000
Apochela Milnesiidae
Genetic Analysis System (Beckman Coulter).
Heterotardigrada Arthrotardigrada Batillipedidae
The sequences were aligned with a previously
published data set (Table 2) (Garey et al., 1999)
according to the secondary structure model (Neefs
et al., 1993). Maximum parsimony (MP) and
Neighbor Joining (NJ) analyses were performed
Echiniscoidea Echiniscidae
using PAUP* and Molecular Evolutionary
Genetic Analysis (MEGA) version 2.0 (Kumar
et al., 2001). The NJ trees were determined using
*From Nelson (2002). the Kimura two-parameter substitution model and

Table 2. List of 18S Ribosomal DNA sequences

Species Length Genbank accesion Reference

Artemia salina 2002 X01723 Nelles et al. (1984)

Placopecten magellanicus 1814 X53899 Rice (1990)
Priapulus caudatus 1814 X80234 Winnepenninckx et al. (1995)
Tenebrio molitor 2083 X07801 Hendriks et al. (1988)
Meloe proscarabaeus 1934 X77786 Chalwatzis et al. (1995)
Okanagana utahensis 1918 U06478 Campbell et al. (1995)
Panulirus argus 1872 U19182 Trapido-Rosenthal et al. (1994)
Echiniscus viridissimus 1824 AF056024 Garey et al. (1996)
Macrobiotus hufelandi 1808 X81442 Giribet et al. (1996)
Macrobiotus tonolli 1735 U32393 Garey et al. (1996)
Hypsibius dujardini 1521 Present study Present study
Milnesium tardigradum 1844 U49909 Aguinaldo et al. (1997)
Calohypsibius schusteri 1384 Present study Present study
Thulinius stephaniae 1686 AF056023 Garey et al. (1996)
Batillipes mirus 1432 Present study Present study

corrected for among-site rate variation. Confi- that Stygarctidae should be the most primitive
dence levels were evaluated by bootstrap analysis. heterotardigrade family. The current analysis
(Fig. 1) suggests instead that Halechiniscidae is
more basal than either Stygarctidae or Renaud-
Results and discussion arctidae and that Oreelidae is the most basal
heterotardigrade. This placement supports earlier
The 50% majority rule tree in Figure 1 was gen- suggestions by Thulin (1928) and Marcus (1929)
erated from an analysis of the morphological data that Echiniscidae is derived from arthrotardi-
set and agrees with the consensus that Eutardi- grades and that Oreellidae should be considered
grada and Heterotardigrada are distinct mono- the most primitive heterotardgrade. The cephalic
phyletic sister groups as evidenced by a high appendages that define heterotardigrades are
bootstrap value of 98%. Among the eutardigrades, present in oreellids, but the dorsal plates, charac-
Eohypsibiidae was a sister group to Macrobioti- teristic of other heterotardigrades, are completely
dae+Hypsibiidae, though the bootstrap support absent. The morphology of the various dorsal
around 60% was minimal. Necopinatidae appears plates appears to provide important characters in
to be basal among the Parachela, which forms a heterotardigrade phylogeny. If the absence of
monophyletic sister group to the Apochela and the dorsal plates represents a secondary loss within the
most basal eutardigrade family, Milnesiidae oreellids, then the basal position of Oreellidae in
(Fig. 1). this study may be an artifact.
Among the heterotardigrade families, boot- The orders Arthrotardigrada and Echiniscoi-
strap values indicate that all branches were dea have been placed as sister groups based on
moderately or highly supported (Fig. 1). Kristen- shared derived characters (Eibye-Jacobsen, 2001).
sen & Higgins (1984) established the family The current results (Fig. 1) suggest that these
Renaudarctidae based on the conclusion that the orders are not sister groups, and it appears that
development of toes and adhesive discs in Arthrotardigrada is paraphyletic containing some
Halechiniscidae and Batillipedidae were derived members of Echiniscoidea.
characters and that the claw insertion in Stygarc- Overall, molecular data agree well with the
tidae was a plesiomorphic state. This assessment morphological-based tree. The NJ and MP trees
agreed with Renaud-Mornant’s (1982) suggestion produced from 18S rRNA gene sequences (Fig. 2)

Figure 1. Phylogeny of tardigrade families with mapped habitat preferences. The analysis supports the monophyletic origin of the class
Eutardigrada and orders Parachela and Apochela along with the class Heterotardigrada; however, the orders within Heterotardigrada
(Arthrotardigrada, Echiniscoidea) may not be monophyletic. Key to Characters: 1. Eutardigrada: No cephalic appendages; lack of
dorsal plates; differentiated placoids; double claws with secondary and primary branches. 2. Heterotardigrada: Cephalic appendages,
digitate legs with or without claws. 3. Parachela: No cephalic papillae; double claws per leg divided into a secondary and primary
branch. 4. Apochela: Six cephalic papillae; two sets of claws per leg, with unbranched primary claw separated from secondary claw; 4–6
peribuccal lamellae. Habitats: Terrestrial (T); Marine (M); or Freshwater (F).

support the morphological character analysis in sequence suggests that heterotardigrades are
indicating that Eutardigrada and Heterotardigrada monophyletic and not likely to be paraphyletic.
are each monophyletic. Within eutardigrades the Mapping of habitat preferences onto the char-
monophyly of Parachela is well supported. Boot- acter tree (Fig. 1) and gene tree (Fig. 3) suggests
strap values for all branches were high with the that tardigrades have adapted to marine environ-
lowest value of 95% for the Hypsibiidae+Macro- ments twice, to freshwater environments at least
biotidae node. The new Calohypsibius sequence three times, and to terrestrial environments twice.
added here indicates that Calohypsibiidae is a sister The placement of the terrestrial Oreellidae as the
group to Hypsibiidae (Fig. 2). However, in the basal heterotardigrade suggests that tardigrades
morphological tree, the relationship between were ancestrally terrestrial. However, the place-
Calohypsibiidae and Hypsibiidae is unresolved ment of Oreellidae in the character tree may be an
(Fig. 1). There is more statistical support for rele- artifact caused by the secondary loss of dorsal
vant nodes in the molecular results (90–100% plates.
bootstrap values) than in the morphological tree Analysis of the morphological data set
(60–64% bootstrap values). The 18S rRNA gene indicates that increased taxon sampling with
sequence from Batillipes mirus (Arthrotardigrada) more detailed characters might prove useful in
adds a second heterotardigrade order to the elucidating the relationships among the tardi-
molecular data set. The analysis of this new grades. Additional 18S rRNA gene sequence data

Figure 2. Tardigrade phylogeny based on l8s rRNA gene sequences. Similar trees were recovered for MP and minimum evolution
analysis. Branch lengths are drawn to scale (substitutions/site). The numbers above each fork are bootstrap values for the NJ tree,
numbers below are the bootstrap values for MP analysis. *Specimens were not identified to species in the original study, but have since
been verified. **The genus Thulinia was recently renamed Thulinius by Bertolani (2003).

Figure 3. Tardigrade molecular topology from Fig 2 with mapped characters and habitat preference. Branches are not to scale, but
bootstrap values are the same as Fig. 2 Key to Characters: 1. Eutardigrada: No cephalic appendages; lack of dorsal plates; differ-
entiated placoids; double claws with secondary and primary branches. 2. Heterotardigrada: Cephalic appendages; digitate legs with or
without claws. 3. Parachela: No cephalic papillae; double claws per leg divided into secondary and primary branch. 4. Apochela: Six
cephalic papillae; two sets of claws per leg, with unbranched primary claw separated from the secondary claw; 4–6 peribuccal lamellae.
5. Thulinius+Hypsibius+Calohypsibius: Claw sequence 2121; Buccal apophyses asymmetric to the frontal plane. 6. Macrobiotus:
Ventral lamina present; claw sequence 2121; 10 perri buccal lamellae. 7. Thulinius+Hypsibius: No ventral lamina. 8. Calohypsibius:
‘‘Calohypsibius type’’ claws with primary and secondary branch fused from base; Ventral lamina absent. 9. Thulinia: peribuccal
lamellae; ‘‘Hybsibius type’’ claws. 10. Hypsibius: Peribuccal lamellae absent; ‘‘Hybsibius type’’ claws. 11. Echiniscus: Armored plates
present. 12. Batillipes: Armored plates absent. Habitats: Terrestrial (T); Marine (M); or Freshwater (F). *Specimens were not identified
to species in the original study, but have since been verified.

will be important in testing the validity of Acknowledgements

morphology based trees and will allow for
combined analyses of molecular and morpho- The authors wish to thank Nigel Marley, Frank
logical data. Sequences from genera within Romano, and Roberto Guidetti for specimen
Oreellidae would be particularly important in samples; Ruth Dewel for her valuable character
determining whether or not the absence of dorsal discussions; Terry Campbell for his comments on
plates represents a secondary loss. the manuscript; and the University of South

Florida Department Of Biology for financial sup- Guidetti, R. & R. Bertolani, 2001. Phylogenetic relationships in
port for PBN. the Macrobiotidae (Tardigrada: Eutardigrada: Parachela).
Zoologischer Anzeiger 240: 371–376.
Jorgensen, A., 2000. Cladistic analysis of the Echiniscidae
Thulin, 1928 (Tardigrada: Heterotardigrada: Echiniscoidea).
Streenstrupia 25: 11–23.
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Schmidt-Rhaesa, A., T. Bartolomaeus, C. Lemburg, U. Ehlers layer. In gastrotrichs the cuticle is brought to the
& J. R. Garey, 1998. The position of the arthropoda in the surface by Golgi vesicles in the form of tiny plates.
phylogenetic system. Journal of Morphology 238: 263–285. (14) Terminal mouth. (15) Cephalic papillae. (16)
Schuster, R. O., D. R. Nelson, A. A. Grigarick & D.
Christenberry, 1980. Systematic criteria of the Eutardi- Cephalic appendages. (17) Peribuccal papillae. (18)
grada. Transactions of the American Microscopical Society Peribuccal lamellae. (19) Buccal tube. (20) Buccal
99: 284–303. tube apophyses. (21) Pharyngeal tube. (22) Pha-
Swofford, D. L., 1998. PAUP. Phylogenetic Analysis Using ryngeal tube apophyses. (23) Ventral lamina. (24)
Parsimony (and Other Methods). Version 4. Sinauer Stylet supports: 0 absent, 1 present, 2 either. (25)
Associates, Sunderland, MA.
Thulin, G., 1928. Ûber die phylogenie und das system der
Placoids: 0 absent, 1 microplacoids only, 2 macro-
tardigraden. Hereditas 11: 207. placoids only, 3 both micro and macroplacoids,
(26) Septulum: 0 absent, 1 present, 2 either. (27)
Claw Structure: 0 absent, 1 single, 2 double sepa-
rated, 3 double connected. (28) Claw sequence,
Appendix 1: List of Morphological Characters separated claws of the heterotardigrades were
scored 1111 and Milnesiidae were scored 1122: 0
(Unless otherwise stated, 0=absent, 1=present) absent, 1 1111, 2 1122, 3 2121, 4 2112. (29) Trans-
(1) Molting by ecdysis. (2) Loss of locomotory verse cuticular bar: 0 absent, 1 present, 2 either. (30)
cilia; Gastrotrichs have locomotory cilia on their Accessory points: 0 absent, 1 present, 2 either. (31)
ventral side and they are covered by the cuticle. (3) Lunulae: 0 absent, 1 present, 2 either. (32) Lateral
Cuticle structure: trilaminate epicuticle, proteina- cirrus A. (33) Median cirrus. (34) Cuticular armor.
ceous exocuticle and chitinous endocuticle; (35) Dorsal segmental plates. (36) Head plate. (37)
gastrotrichs do not have a chitinous endocuticle. (4) Median plate 1. (38) Median plate 2. (39) Median
Parthenogenesis. (5) Circumpharyngeal nerve ring. plate 3. (40) Caudal plate. (41) Pseudosegmental
(6) Complete gut. (7) Permanent genital pore sep- plate: 0 absent, 1 present, 2 either. (42) Peduncles.
arate from anus. (8) Adhesive glands. (9) Protone- (43) Clava. (44) Digitate legs. (45) Leg 4 morphol-
phridia. (10) Adult gut functional. (11) Triangular ogy: 0 absent, 1 spine, 2 papilla. (46) Eyespots: 0
shaped pharynx. (12) Stylets. (13) Formation of absent, 1 present, 2 either. (47) Cloaca. (48) Sexual
epicuticle: appears over the tip of short microvilli in dimorphism of claws (49) Sexual dimorphism of the
patches that laterally merge to form a continuous gonopore. (50) Pharyngeal stripes.

Appendix 2: Morphological Data Matrix