Toward a better understanding of the phylogeny of the

Asplanchnidae (Rotifera)

Elizabeth J. Walsh1,*, Robert L. Wallace2 & Russell J. Shiel31Department of Biological Sciences, University of Texas at El Paso, El Paso, TX 79968, USA2Department of Biology, Ripon College, Ripon, WI 54971, USA3The University of Adelaide, Adelaide, Australia(*Author for correspondence: E-mail: ewalsh@utep.edu)

Key words: cladistics, evolution, nrDNA, mtDNA, morphology, Monogononta, Synchaetidae

Abstract

We investigated the phylogenetic relationships of Family Asplanchnidae using both morphological andmolecular data. The morphological database, comprising 23 characters from 19 taxa (15 Asplanchnidaeand 4 outgroups), was compiled from a survey of the literature and our own observations; the moleculardata (ITS and V4 region nuclear regions and mitochondrial cox1) was sequenced from specimens that wecollected. Our analysis of the morphological data set (maximum parsimony) yielded 12 most-parsimonioustrees with a tree length of 27 steps. From this analysis we conclude (1) Asplanchnidae is a monophyleticgroup as are the three genera comprising it, (2) there is no compelling support for the argument thatAsplanchna should be separated into two discrete genera, and (3) there is some support for the proposal thatAsplanchnidae and Synchaetidae are sister groups. Our analysis of the molecular data set supports the firsttwo of these conclusions while the sister group of the family varied depending on the gene region analyzedand families and genera included. Current understanding of the phylogeny of Asplanchnidae is hamperedby the need for additional informative morphological characters and a lack of molecular data for the genusHarringia and several other members of the Asplanchnidae.

Introduction

Family Asplanchnidae (Eurotatoria; Monog-ononta; Ploima) comprises 15 species ofomnivorous rotifers, assigned to three genera(Ruttner-Kolisko, 1974; Koste, 1978; Jose dePaggi, 2002; Segers, 2002). These genera havespecific habitat preferences that vary along a gra-dient from benthic to strictly planktonic. Thebenthic genus Harringia has a well-developed footand is usually associated with plants; the semi-pelagic genus Asplanchnopus has a reduced footand can be found with plants; the planktonic genusAsplanchna lacks a foot and is typically limited toopen water. Whereas Harringia has a completedigestive system, the other two genera lack a gut;

the etymon of both genera (G. a, lacking + G.,splanchnum, the inward parts) refers to this fea-ture. Although much is known about the generalbiology of Asplanchnidae (e.g., Salt et al., 1978;Gilbert et al., 1979; Gilbert 1980, 1999; Joanido-poulos & Marwan, 1998, 1999; Kappes et al.,2000), we are aware of only two specific hypothe-ses regarding its phylogeny and neither has beenexplored (Sudzuki, 1964; Kutikova, 1983). Thesehypotheses were formulated using the principles ofevolutionary taxonomy (Ridley, 1986).

Sudzuki�s (1964: Fig. 2 & 75–78) genus-levelphylogeny separates Asplanchna into Asplanchnaand Asplanchnella based on vitellarium morphol-ogy: Asplanchna = globular ovarium (sic) [vitel-larium] (i.e., priodonta, herrickii); Asplanchnella

Hydrobiologia (2005) 546:71–80 � Springer 2005A. Herzig, R.D. Gulati, C.D. Jersabek & L. May (eds.) Rotifera X: Rotifer Research: Trends, New Tools and Recent AdvancesDOI 10.1007/s10750-005-4103-8

= elongated vitellarium (i.e., brightwellii, girodi,intermedia, sieboldii). He also emphasized thenumber of nuclei of the vitellarium in his key (seealso Gilbert et al., 1979). Since Sudzuki�s contri-bution, a third vitellarium shape has beendescribed (sacciform, not spherical; Koste & To-bias, 1989), but no one has wedded this observa-tion into his scheme. Both Ruttner-Kolisko (1974:100) and Koste (1978: 449) support Sudzuki�s po-sition by subdividing Asplanchna based on vitel-larium shape, but neither examined the suitabilityof this separation. However, in her taxonomictreatise Jose de Paggi (2002) retained Asplanchna(sensu stricto) without accepting Sudzuki�s parti-tion. Her assessment was based on the fact thatvitellarium shape varies in other Asplanchnidae(i.e., Asplanchnopus), thus suggesting that vitel-larium shape is too unreliable to warrant its use asa critical character for taxonomy within this fam-ily. This same point can be made for the nuclearnumber in the gastric and yolk glands and thenumber of flame cells in the protonephridia.The second phylogenetic hypothesis published onthe Asplanchnidae was that of Kutikova (1983:Fig. 2). She argued for a sister relationship withSynchaetidae based on the details of coronalmorphology and function.

Thus, our purpose was to investigate the phy-logeny of the Asplanchnidae and specifically toexamine the hypotheses of Sudzuki and Kutikova.To do this, we performed cladistical analyses onmorphological and molecular databases.

Materials and methods

Morphological data

Based on a survey of the literature and our ownobservations, a data matrix of 23 characters on 19monogonont rotifers comprising the 15 recognizedspecies of Asplanchnidae and four outgroupsBrachionus (Brachionidae), Polyarthra and Syn-chaeta (Synchaetidae), and Trichocerca (Trichoc-ercidae) was constructed (Table 1). For the presentanalysis, we followed the general protocol used byMelone et al. (1998) in their study of rotiferanmorphology.

The program PAUP* (4.0b10; Swofford, 2002)was used for all searches on the data matrix. A

heuristic search strategy was employed in whichthe ancestral states were considered to be unrootedand the transformation types unordered. We ransearches first without and then with the charactersvitellarium shape, cerebral eyespot, and ramusdentition. We also applied increased weight to allcombinations of the following five characters(Foot, Pedal gland, Gut, Trophi type, Femalepolymorphism). Tree statistics reported here are(1) Tree Length (TL), (2) Consistency Index (CI)(excluding uninformative characters), and (3)Retention Index (RI). The reconstructions re-ported for the analysis include the two characteroptimization algorithms available in PAUP forrooted trees (ACCTRAN, DELTRAN). As a testof robustness we ran a bootstrap simulation(n = 103 replications) with and without includingthe characters vitellarium shape, cerebral eyespot,and ramus dentition. We also employed the per-mutation tail probability (PTP) test (Faith &Cranston, 1991) to examine cladistic structure ofthe data set again without the three charactersnoted above. Standard constraint methodologieswere used to generate tree statistics for the alter-native tree topologies reported as optimized onour data matrix.

Molecular data: sampling and processing

Species used for the present studywere collected andanalyzed from multiple sites as shown in Table 2.When possible, rotifers were starved before preser-vation and subsequent extraction of DNA. Allrotifers were identified to species using keys ofKoste (1978) and Jose de Paggi (2002). Voucherspecimens of all species were preserved in 70%EtOH and are stored at Laboratory for Environ-mental Biology (UTEP). DNA was isolated andamplified from axenized fresh or ethanol preservedanimals using Chelex-100 (Bio-Rad) as describedinWalsh&DeLaRiva (unpublished). Primers usedto amplify the 690 bpmitochondrial cox1 gene wereLCO1490 (5¢-GGTCAACAAATCATAAAGAT-ATTGG-3¢) and HCO2198 (5¢-TAAACTTCA-GGGTGACCAAAAAATCA-3¢) (Folmer et al.,1994), and those for the nuclear ribosomal generegions were those for the 18S variable regionV4 (V4A: 5¢-TGCAGTTAAAAAGCTCGTAGT-3¢, V4B: 5¢-CCCTTCCGTCAATTCCTTTAAG-3¢)and internal transcriber spacer region (ITS4:

72

5¢-TCCTCCGCTTATTGATATGC-3¢, ITS5: 5¢-GGAAGTAAAAGTCGTAACAAGG-3¢; Whiteet al., 1990). The ITS primers amplify ITS1, 5.8S,and ITS2 regions of the 18S nuclear ribosomalgene complex. All amplifications included a nega-tive control. Amplification products were exam-ined by electrophoresis to verify their size andcleaned with GeneClean kits (Bio101) beforesequencing. Amplified products were sequenceddirectly using SequiTherm Excel LI kits (EpiCen-tre Technologies) and run on a LI-COR 4200Series automated sequencer. All gene regions orgenes were sequenced at least twice in both direc-tions. Sequences were aligned using Clustal W(Thompson et al., 1994)

Phylogenetic trees of morphological characterswere constructed using maximum parsimony (MP),and maximum likelihood (ML) algorithms, imple-mented in PAUP* (Swofford, 2002). All searcheswere heuristic, with TBR branch swapping and theMULPARS option in effect. For MP and MLsearches, taxa were added randomly, with 10 addi-tion-sequence replicates. To assess support fornodes in the MP and ML trees, we used non-para-metric bootstrapping (Felsenstein, 1985). Neigh-bor-joining was used to construct all molecularphylogenies using uncorrected �p� distances.

In the molecular analysis, in contrast to themorphological analysis, we do not include somespecies due to the lack of availability of samples.

Table 1. Data matrix of 23 characters used in the morphological analysis. Outgroups, Brachionus, Polyarthra, Synchaeta, and

Trichocerca; ingroups = 9 Asplanchna (As), 4 Asplanchnopus (An), and 2 Harringia (H)1

Taxa Characters

1–4 5–8 9–12 13–16 17–20 –23

Brachionus 0000 0010 0000 0000 0000 110

Polyarthra 1000 0000 0010 0000 0001 020

Synchaeta 0000 0110 0011 0000 0000 020

Trichocerca 0000 1000 0010 0000 1010 110

As. brightwellii 1111 0002 0120 1101 0100 021

As. girodi 1111 0001 0120 1100 0000 020

As. priodonta 1111 0000 0120 1100 0000 020

As. herrickii 1111 0000 0120 1100 0000 020

As. intermedia 1111 0001 0120 1111 0000 021

As. sieboldii 1111 0002 0120 1101 0000 021

As. silvestrii 1111 0002 0120 1100 0000 021

As. tropica2 1111 0000 0120 1100 0000 02?

As. asymmetrica3 1111 0002 0120 1101 1000 02?

An. dahlgreni 0011 0000 1020 1100 0000 010

An. hyalinus 0011 0001 0020 1111 0000 010

An. multiceps 0011 0002 0020 1110 0000 010

An. bhimavaramensis 0011 0002 ?020 1100 0000 010

H. eupoda 0001 0001 1?20 0000 0000 000

H. rousseleti 0001 0001 0?20 0000 0000 000

1This data set was derived from the reviews of Koste (1978), Jose de Paggi (2002), Hollowday (2002), original literature as available to

us (Hudson, 1886, in Hudson & Gosse, 1886; Harring, 1913; Myers, 1934; Dhanapathi, 1975; Gilbert et al., 1979; Shiel & Koste, 1985;

Koste & Tobias, 1989), and our own observations. Character states correspond to the descriptions provided in the Appendix (missing

data = ?). NB: all Asplanchnopus species are oviparous (cf. Jose de Paggi, 2002); spellings of certain Asplanchna species have been

corrected according to Jose de Paggi (2002). 2As. tropica is known only from preserved material, thus the status of character #23

(Female polymorphism; i.e., the production of body wall outgrowths) is unknown. 3As. asymmetrica has been observed alive by one of

us (RJS) on several occasions (Ryan�s Billabong, Australia). While none of these observations indicated the presence of body wall

outgrowths, we coded the status of character #23 as unknown (=?) on the basis of its morphological similarities to As. brightwellii.

However, additional cladistic analyses were run with a code of 0 for this character.

73

This includes the genus Harringia, which has onlybeen reported 3 times in the Zoological Recordsdating back to 1970. Other species that need to beincluded in future analyses are A. intermedia,A. asymmetrica, A. sylvestrii and A. tropica.

Results

Morphological analysis

Naturally because we ran our searches with thevariations of excluding some characters and re-weighting others, our study yielded several trees.Of all the analyses the ones in which the charac-teristics of vitellarium shape, cerebral eyespot, andramus dentition were eliminated yielded the mostinteresting results; here we present an interpreta-tion of that analysis. This search strategy yielded12, equally most-parsimonious trees (TL = 27,CI = 0.746, RI = 0.915) (Fig. 1). However, withinclusion of any combination of the three charac-ters noted above the topology of the trees gener-ated remained fundamentally the same, but withmany additional steps (TL ‡29) as well as addi-tional polytomies.

Percentages of bootstrap replicates (n = 103)supporting nodes of the most-parsimonious treeare reported in Figure 1. None of the PTP testsyielded a tree with a TL equal to or with fewersteps than were otherwise obtained by any of thesearch strategies. Thus the null hypothesis, that thedata defining these trees show no cladistic struc-ture, is rejected (p � 0.01).

We used constraint analysis to examine thephylogenies of Sudzuki and Kutikova fixed on ourdata matrix, without the characters vitellariumshape, cerebral eyespot, and ramus dentition.Kutikova�s hypothesis proved to be congruent toour tree with the same number of steps but yield-ing only six trees. We found similar results whenwe added the three characters noted above in allpossible combinations, but again sometimes withadditional steps and polytomies. However, Sud-zuki�s hypothesis possessed two more steps thanour phylogeny. Again we had similar results whenwe added the three characters noted above in allpossible combinations. Based on our morpholog-ical analysis we conform with Jose de Paggi�s(2002) conclusion that Sudzuki�s (1964) proposalto separate Asplanchna into two genera is notwarranted. In fact, it makes more sense to group

Table 2. Species list and localities in which they were collected for molecular analysis

Species Location

Ingroup

Asplanchna brightwellii Lake Carnegie, Princeton, NJ, USA

Asplanchna brightwellii Lake Eyre Basin, AUS

Asplanchna girodi Ascarate Pond, El Paso, TX, USA

Asplanchna priodonta Lake Naomi, Pocono Pines, PA, USA

Asplanchna priodonta Diamantina River, Lake Eyre Basin, AUS

Asplanchna herrickii Lake Sagatan, Collegeville, MN, USA

Asplanchna intermedia Hueco Tanks State Historic Site, El Paso, TX, USA

Asplanchna sieboldii Several sites near El Paso, TX, USA

Asplanchna sp. Kangeroo Island, AUS

Asplanchnopus hyalinus Hueco Tanks State Historic Site, El Paso, TX, USA

Asplanchnopus multiceps Hueco Tanks State Historic Site, El Paso, TX, USA

Asplanchnopus multiceps Pelican Pond, UT, USA

Outgroup

Trichocerca rattus Hueco Tanks State Historic Site, El Paso, TX, USA

Synchaeta pectinata Lake Naomi, Pocono Pines, PA, USA

Synchaeta littoralis Elephant Butte Reservoir, NM, USA

Polyarthra dolichoptera Elephant Butte Reservoir, NM, USA

74

members of this genus based on their propensityfor female polymorphism (see Gilbert et al., 1979).

Molecular analysis

Analyses of all gene regions (except cox1) supportthe monophyly of the Asplanchnidae (Figs. 2–5).Analysis of the V4 region shows the sister group to

Asplanchnidae as a clade represented by Brachi-onidae and Polyartha and Trichocerca. Since alltaxa are not represented in phylogenies derivedfrom the other gene regions, the molecular dataare inconclusive at this time. Analyses of the ITSregions and cox1 resolve relationships at the spe-cies level. In the ITS1 analysis (Fig. 3), the epi-pelagic Asplanchnopus grouped with planktonic

Figure 1. A phylogeny of Family Asplanchnidae based on morphological characters. This 50% majority-rule, consensus tree was

generated using an unrooted and unordered search strategy that excluded the following three characters from our data matrix

(Table 1): Vitellarium (#8), Cerebral eyespot (#9), and Ramus dentition (#15). Because some characters were uninformative, not all of

the characters contributed to this tree. Numbers in bold print (1–10) refer to internal nodes. Numbers in italics below each node are the

percentages of 1000 bootstrap replicates supporting that portion of the tree. Membership in Sudzuki�s (1964) Asplanchnella

(= elongated vitellarium) indicated by a * or a + (described after 1964). Changes in character state for the tree that are supported by

both character optimization algorithms (ACCTRAN, DELTRAN) are reported below. These changes are annotated by node number

or terminal taxon (in bold print). Brachionus. Coronal bristles: present (#7); Trophus: malleate (#11). (1) Trichocerca. Coronal palps:

present (#5); Trophus: asymmetrical (#17); Body: asymmetrical (#19). (2) Lorica: illoricate (#21). Synchaetidae – Polyarthra. Foot:

absent (#1); Paddles: present (#20). Synchaeta. Coronal auricles: present (#6); Coronal bristles: present (#7); Hypopharynx: present

(#12). (3) Asplanchnidae: Coronal ciliation: single wreath (#4); Trophus: incudate (#11). (4) Harringia: Cerebral eyespot (#9) (dropped

in our standard analysis). (5) Gut: incomplete (#3); Manubrium function: reduced/vestigal (#13); Unci function: reduced/vestigal

(#14). (6) Asplanchnopus: Vitellarium Shape (#8) (dropped in our standard analysis); Cerebral eyespot (#9) (dropped in our standard

analysis); Ramus dentition (#15) (dropped in our standard analysis); Apophysis-subapophysis (#16); other autapomorphies. (7)

Asplanchna. Foot: absent (#1); Pedal gland: absent (#2); Amictic egg development: oviviviparous (#10). (8 & 9) Apophysis-sub-

apophysis absent (#16). (10) Apophysis-subapophysis present on trophus (#16).

75

Asplanchna girodi, this is most likely a problem ofincomplete taxon sampling since in all otheranalyses the Asplanchnopus are the sister group toAsplanchna. The ITS2 analysis reflects the eco-logical niches of rotifers as does the cox1 analysis(Figs. 4, 5).

Discussion

Based on both the morphological analysis andpreliminary molecular analyses, we conclude thefollowing: (1) Asplanchnidae is a monophyleticgroup, as are the three genera that comprise it; (2)there is no compelling support for the argumentthat Asplanchna should be separated into twodiscrete genera; (3) there is some support for theproposal that Asplanchnidae and Synchaetidae aresister groups. Unfortunately, using morphologicalcharacters, species relationships within genera

were not well resolved. To aid in that resolution,improvements in the morphological dataset shouldbe made. These refinements might include deter-mining whether A. asymmetrica and A. tropica arecapable of female polymorphism (i.e., producingbody wall outgrowths) and establishing categoriesthat distinguish among the extent to which bodywall outgrowths develop.

The molecular analysis is promising in that forthose taxa represented, species relationships wereresolved. The utility of these gene regions inresolving phylogenies has been demonstrated formany organisms and recently in the brachionidrotifers (Gomez et al., 2002; Derry et al., 2003) (seepapers in this volume Part I, Phylogeny andEvolution + II, Genetics and Molecular Ecol-ogy). We have found that a combination of generegions works best to resolve family, generic andspecies relationships. The V4 nuclear region workswell at the family level but poorly at the species

Figure 2. A Neighbor-Joining analysis of Family Asplanchnidae based on the V4 variable region of the 18S nuclear ribosomal gene.

Brachionidae sequences were extracted from Walsh & De La Riva (unpublished). Rhinoglena sequence was extracted from Segers &

Walsh (in prep.).

76

Figure 3. A Neighbor-Joining analysis of Family Asplanchnidae based on the internal transcribed spacer region 1 of the 18S nuclear

ribosomal gene complex.

Figure 4. A Neighbor-Joining analysis of Family Asplanchnidae based on the internal transcribed spacer region 2 of the 18S nuclear

ribosomal gene complex.

77

level (Walsh & De La Riva, submitted) whilethe reverse is true for the cox1 and ITS regions(Gomez et al., 2004). Future work will focus onobtaining complete sequence data for the remain-ing members of the Asplanchnidae.

To our knowledge this is the first time that bothmorphological and molecular data have been em-ployed in a cladistic analysis of a rotiferan family.In this regard we have taken up the challengeposed by Melone et al. (1998) who argued thatshould morphological and molecular studies re-main firmly compartmentalized our understandingof rotifer phylogeny would be unobtainable.Unfortunately, significant gaps in our knowledgeof the Asplanchnidae remain and this deficiencyhampers our comprehension of their evolution.

Acknowledgements

The following researchers aided us in this study:B. J. Dingmann (MNSP Gallagher Fellow),A. Armendariz, M. Fout, and M. Ortega(Asplanchnidae); V. De La Riva (Brachionidae),and A. Frias (Synchaeta, Polyarthra). ElizabethWurdak kindly provided Cisplanchna for analyses.We thank J. J. Gilbert, C. D. Jersabek, T. Schro-der, and an anonymous reviewer who providedvaluable comments on our analysis. We also thankthe staff at Hueco Tanks State Historic Site (col-lecting permits #66–99; 07–02). This research wassupported by NSF HRD-9628568 and NIH5G12RR008124. We also thank the Ripon College

Figure 5. A Neighbor-Joining analysis of Family Asplanchnidae based on the mitochondrial cox1 gene. Brachionus sequences are from

Gilbert & Walsh, this volume. Rhinoglena and Epiphanes sequences are from Segers & Walsh (in prep.).

78

(RLW) and University of Texas at El Paso (EJW)for additional funding.

References

Derry, A.M., P. D.N.Hebert & E. E. Prepas, 2003. Evolution of

rotifers in saline and subsaline lakes: A molecular phyloge-

netic approach. Limnology and Oceanography 48: 675–685.

Dhanapathi, M. V. S. S., 1975. Rotifers from Andhra Pradesh,

India. Zoological Journal of the Linnean Society 57: 85–94.

Faith, D. P. & P. S. Cranston, 1991. Could a cladogram this

short have arisen by chance alone? On permutation tests for

cladistic structure. Cladistics 7: 1–28.

Felsenstein, J., 1985. Confidence limits on phylogenies: An

approach using the bootstrap. Evolution 39: 783–791.

Folmer, O., M. Black, W. Hoeh, R. Lutz & R. Vrijenhoek,

1994. DNA primers for amplification of mitochondrial

cytochrome c oxidase subunit 1 from diverse metazoan

invertebrates. Molecular Marine Biology and Biotechnology

3: 294–299.

Gilbert, J. J., 1980. Feeding in the rotifer Asplanchna: behavior,

cannibalism, selectivity, prey defenses, and impact on rotifer

communities. In Kerfoot, W. C. (ed.), Evolution and Ecol-

ogy of Zooplankton Communities. Univ. Press New England,

Hanover, NH, 158–172.

Gilbert, J. J., 1999. Kairomone-induced morphological defenses

in rotifers. In Tollrian, R. & C. D. Harvell (eds), The Ecol-

ogy and Evolution of Inducible Defenses. Princeton Uni-

versity Press, Princeton, NJ, 127–141.

Gilbert, J. J., C. W. Birky & E. S. Wurdak, 1979. Taxonomic

relationships of Asplanchna brightwelli, A. intermedia, and

A. sieboldi. Archiv fur Hydrobiologie 87: 224–242.

Gomez, A., M. S. Serra, G. R. Carvalho & D. L. Hunt, 2002.

Speciation in ancient cryptic species complexes. Evidence

from molecular phylogeny of Brachionus plicatilis (Rotifera).

Evolution 56: 1431–1444.

Harring, H. K., 1913. A list of the Rotatoria of Washington and

vicinity with descriptions of a new genus and ten new species.

Proceedings of the U.S. Natural Museum 46: 387–405.

Hollowday, E. D., 2002. Rotifera, Vol. 6. Family Synchaetidae.

In Nogrady, T. & H. Segers (eds), Guides to the Identifica-

tion of the Microinvertebrates of the Continental Waters of

the World. Backhuys Publishers, The Hague, 87–211.

Hudson, C. T. & P. H. Gosse, 1886. The Rotifera or Wheel An-

imacules. Vol. I. Longmans, Green, and Co. London, 128 pp.

Joanidopoulos, K. D. &W.Marwan, 1998. Specific behavioural

responses triggered by identified mechanosensory receptor

cells in the apical field of the giant rotifer Asplanchna sieboldi.

Journal of Experimental Biology 201: 169–177.

Joanidopoulos, K. D. & W. Marwan, 1999. A combination of

chemosensory and mechanosensory stimuli triggers the male

mating response in the giant rotifer Asplanchna sieboldi.

Ethology 105: 465–475.

Jose de Paggi, S., 2002. Rotifera, Vol. 6. Family Asplanchnidae.

In Nogrady, T. & H. Segers (eds), Guides to the Identifica-

tion of the Microinvertebrates of the Continental Waters of

the World. Backhuys Publishers, The Hague, 1–27.

Kappes, H., C. Mechenich & U. Sinsch, 2000. Long-term

dynamics of Asplanchna priodonta in Lake Windsborn with

comments on the diet. Hydrobiologia 432: 91–100.

Koste, W., 1978. Rotatoria. Die Radertiere Mitteleuropas. 2

volumes. Gebruder Borntraeger, Berlin, Stuttgart, Germany,

Textband 673 pp., Tafelband 234 Tafeln.

Koste, W. & W. Tobias, 1989. Rotatorien der Selingue-Tal-

sperre in Mali, Westafrika (Aschelminthes). Senckenbergi-

ana biologica 69: 441–466.

Kutikova, L. A., 1983. Parallelism in the evolution of rotifers.

Hydrobiologia 104: 3–7.

Melone, G., C. Ricci, H. Segers & R. L. Wallace, 1998. Phy-

logenetic relationships of phylum Rotifera with emphasis on

the families of Bdelloidea. Hydrobiologia 387/388: 101–107.

Myers, F. J., 1934. The distribution of Rotifera on Mount

Desert Island, Part V. A new species of Synchaetidae and

new species of Asplanchnidae,Trichocercidae and Brachi-

onidae. American Museum Novitates 700: 1–16.

Ridley, M., 1986. Evolution and classification: the reformation

of cladism. Longman, NY, 201 pp.

Ruttner-Kolisko, A., 1974. Planktonic Rotifers: biology and

taxonomy. Die Binnengewasser (Supplement) 26: 1–146.

Salt, G. W., G. F. Sabbadini & M. L. Commins, 1978. Trophi

morphology relative to food habits in six species of rotifers

(Asplanchnidae). Transactions of the American Microscopi-

cal Society 97: 469–485.

Segers, H., 2002. The nomenclature of the Rotifera: annotated

checklist of valid family- and genus-group names. Journal of

Natural History 36: 631–640.

Shiel, R. J. & W. Koste, 1985. New species and new records of

Rotifera (Aschelminthes) from Australian waters. Transac-

tion of the Royal Society of Australia 109: 1–15.

Sudzuki, M., 1964. New systematical approach to the Japanese

planktonic Rotatoria. Hydrobiologia 23: 1–124.

Swofford, D. L., 2002. PAUP* - Phylogenetic Analysis Using

Parsimony (* and Other Methods). Ver. 4 [Computer soft-

ware]. Sinauer Associates, Sunderland, MA [with periodic

on-line updates].

Thompson, J. D., D. G. Higgins & T. J. Gibson, 1994.

ClustalW: Improving the sensitivity of progressive multiple

sequence alignment through sequence weighting, position-

specific gap penalties and weight matrix choice. Nucleic

Acids Research 22: 4676–4680.

White, T. J., J. Bruns, S. Lee, & J. Taylor, 1990. Amplification

and direct sequencing of fungal ribosomal RNA genes for

phylogenetics. In Innis, M., D. Gelfand, J. Sninsky & T.

White (eds), PCR Protocols: A Guide to Methods and

Applications. Academic Press, Inc, San Diego.

Appendix

Character descriptions (unless otherwise defined,p, present, a, absent).

1. Foot. [p = 0, a = 1]. 2. Pedal Gland [p = 0,reduced/absent = 1] (NB: As. herrickii rudimen-tary pedal gland = 1). 3. Gut. [complete = 0,

79

incomplete = 1]. 4. Coronal Ciliation [(0 = cir-cumapical bands, 1 = single wreath)]. 5. CoronalPalps [a = 0, p = 1]. 6. Coronal Auricles [a = 0,p = 1]. 7. Coronal Bristles [a=0, p=1]. 8. Vitel-larium Shape. [spherical/sacciform=0, elon-gate=1] (dropped in our standard analysis). 9.Cerebral Eyespot. [p = 0, a = 1] (dropped in ourstandard analysis). 10. Amictic Egg Development.[oviparous = 0, ovovivparous = 1]. 11. Trophi.[malleate = 0, virgate = 1, incudate = 2]. 12.Hypopharynx [a = 0, p = 1]. 13. ManubriaFunction. [functional = 0, reduced/vestigal = 1].

14. Unci Function. [functional = 0, reduced/vesti-gial = 1]. 15. Ramus denticulation. [p = 0, a = 1](dropped in our standard analysis). 16. Apophysis-subapophysis (on trophus). [a = 0, p = 1]. 17.Trophus Symmetry. [symmetrical = 0, asymmet-rical = 1]. 18. Lamella (behind rami apices).[a = 0, p = 1]. 19. Body Symmetry [0 = sym-metrical, 1 = asymmetrical]. 20. Paddles. [a = 0,p = 1]. 21. Lorica [a = 0, p = 1]. 22. Habitat.[benthic = 0, semipelagic/littoral = 1, plank-tonic = 2]. 23. Female polymorphism (body walloutgrowths). [a = 0, p = 1].

80