Diversity of the genus Daphniopsis in the salinewaters of Australia

Paul D.N. Hebert and Christopher C. Wilson

Abstract: Although members of the cladoceran genusDaphniopsisform a dominant element of the fauna in the salineinland waters of Australia, their taxonomy has been in flux. In this study allozyme analysis was employed to examinethe diversity, distributions, and reproductive biology of species in this genus. The results establish thatD. pusilla, aspecies formerly thought to be widespread, is restricted to Western Australia, while a newly described species,D. truncata, which shares the attribute of producing a one-egged ephippium, is broadly distributed. The results of thisstudy verify the taxonomic validity of the three recognized species ofDaphniopsis, which produce two-egged ephippia,but another member of this group,D. wardi, is described from Western Australia. All populations were found to repro-duce by cyclic parthenogenesis, except for one obligately asexual population ofD. pusilla × D. truncatahybrids. Noother case of hybridization was detected, although two species co-occurred in 15% of habitats. The six species ofDaphniopsisnow known from Australia appear to represent another example of an endemic radiation in the salinelakes of this continent.

Résumé: Bien que les cladocères du genreDaphniopsisconstituent un élément dominant de la faune des eaux salinesà l’intérieur de l’Australie, leur taxonomie est changeante. Nous avons utilisé des allozymes pour étudier la diversité, larépartition et la biologie de la reproduction chez les espèces de ce genre. Les résultats indiquent queD. pusilla, uneespèce réputée répandue, est en fait restreinte à l’Australie occidentle, alors que l’espèce nouvellement décrite,D. trun-cata, qui possède également un éphippium à un seul oeuf, a une vaste répartition. Nous avons examiné la validité taxo-nomique des trois espèces reconnues deDaphniopsisqui produisent des éphippiums à deux oeufs et décrivons uneautre espèce du même groupe,D. wardi, de l’Australie occidentale. Toute les populations étudiées se reproduisent parparthénogenèse cyclique, à l’exception dune population asexuée obligée d’hybrides deD. pusilla × D. truncata. Nousn’avons pas observé d’autres cas d’hybridation, bien que deux espèces aient été trouvées en cohabitation dans 15 %des habitats utilisés. Les six espèces deDaphniopsisd’Australie semblent constituer un autre cas de radiation endé-mique dans les lacs salés du continent.

[Traduit par la Rédaction] 808

Hebert and WilsonIntroduction

Although few cladoceran species occur in saline waters,most species ofDaphniopsisare restricted to these settings.Until recently it was thought that their taxonomic diversitywas low, with only a single species reported from each offive continents: Australia, South America (Hann 1986),North America (Schwartz and Hebert 1985), Antarctica(Laybourn-Parry and Marchant 1992), and Asia (Sars 1903).Serventy (1928–1929) provided the first record ofDaphniopsisfrom Australia, describingD. pusilla from the saline pondsof Rottnest Island. For more than 50 years, all Australiancollections ofDaphniopsiswere identified as this species,which was consequently thought to have a broad distribution

(Sergeev and Williams 1984). However, Kokkinn and Wil-liams (1987) suggested that taxonomic diversity was higher,noting the occurrence of six morphologically divergent typesof Daphniopsisephippia in Australia. The differences in ephip-pial morphology that they reported were not subtle: two ofthe variants contained a single egg chamber, as inD. pusilla,while the others had two chambers.

Subsequent morphological studies led to the recognitionof three new species ofDaphniopsisproducing two-eggedephippia.Daphniopsis australiswas described from Tas-mania (Sergeev and Williams 1985), but also occurs in thesaline lakes of Victoria, whereD. quadrangulais endemic(Sergeev 1990a). Daphniopsis queenslandensiswas describedfrom Lake Wyara in southwestern Queensland (Sergeev1990b, 1994), but was later shown to occur in nearby salinelakes in New South Wales (Timms 1993). These taxa,together withD. pusilla, produce ephippia resembling fourof the six types reported by Kokkinn and Williams (1987),suggesting that at least two species remain undescribed.

Past work on theDaphniopsisfauna of Australia has pro-gressed little beyond taxonomic description and, as alreadynoted, even this task is not yet complete. The aim of thepresent study was to provide a more thorough understandingof taxonomic diversity and species distributions by carryingout allozyme analyses on populations from South Australia

Can. J. Zool.78: 794–808 (2000) © 2000 NRC Canada

794

Received June 23, 1999. Accepted November 25, 1999.

P.D.N. Hebert.1 Department of Zoology, University ofGuelph, Guelph, ON N1G 2W1, Canada.C.C. Wilson.2 Research School of Biological Sciences,Australian National University, Canberra, A.C.T. 2601,Australia.

1Author to whom all correspondence should be addressed(e-mail: phebert@uoguelph.ca).

2Present address: Ministry of Natural Resources, TrentUniversity, Peterborough, ON K9J 8N8, Canada.

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and Western Australia, the states with the highest density ofsaline habitats (DeDeckker 1983). The allozyme analyseswere also used to assess breeding systems, compare levels ofgenetic variation among species, and determine if inter-specific hybridization occurred between the taxa.

Materials and methods

CollectionsCollections ofDaphniopsisspp. were made from 20 habitats in

Western Australia in October 1995 and from 35 South Australianhabitats in November 1996 (Fig. 1, Table 1). The collections fromWestern Australia included three populations from Rottnest Island,the type locality forD. pusilla. Reference collections of the otherthree species were also obtained from their type locality or a habi-tat in close proximity to it.Daphniopsis queenslandensiswas ob-tained from its type locality (Lake Wyara) and a sample ofD. quadrangulawas obtained from a lake near its type locality inVictoria. As the Tasmanian type locality forD. australis lackedwater throughout 1995 and 1996, a collection of this species was

obtained from a habitat near Colac, Victoria, described by Sergeevand Williams (1985).

Allozyme analysesFollowing their collection, individuals were placed in deionized

water for 10–15 min prior to cryopreservation, as pilot studiesshowed that allozyme resolution was impeded by the salt waterthat, otherwise, was trapped within their carapaces. Allozyme phe-notypes were determined for 20 individuals from each populationat 10 loci by means of cellulose acetate electrophoresis (Hebertand Beaton 1993). The following loci were examined: aldehyde oxi-dase (Ao), amino aspartate transferase (Aat), arginine phospho-kinase (Apk), fumarate hydratase (Fum), glucose-6-phosphateisomerase (Gpi), lactate dehydrogenase (Ldh), malate dehydro-genase (Mdh), malic dehydrogenase (Me), mannose-6-phosphateisomerase (Mpi), and phosphoglucomutase (Pgm). All allozymedata were analyzed using BIOSYS-1 version 1.7 (Swoffordand Selander 1989).

Genetic characterization of single populationsThe genotypic structure of each population (Hebert et al. 1988)

was summarized by calculating three parameters: the genotypic

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Fig. 1. Distribution records for 6Daphniopsisspecies in South Australia and Western Australia.

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diversity ratio (GDR), the probability of the genotypic array as-suming Hardy–Weinberg (HW) equilibrium, and the directionalityof deviations from HW equilibrium (F). The last parameter, the in-breeding coefficient (Fis), was used to indicate the extent ofheterozygote excess or deficit, an important indicator in organismsthat reproduce asexually. The proximity of genotypic-frequencydistributions at individual loci to HW equilibrium was summarizedfor each population by calculating the average of the log-transformed probability of the genotypic arrays at each polymor-phic locus. The GDR summarizes the relationship between theobserved number of multilocus genotypes encountered in theallozyme analysis and the average expected number of genotypesin a sexual population with the same gene frequencies generatedfrom 100 Monte Carlo simulations. The GDR approximates unityin populations that show linkage equilibrium and conformity toHW expectations. The probability that the observed number of ge-notypes differed from that expected in a sexually reproducing pop-ulation was assessed using at test of single observations againstthe mean and variance generated from the simulations (Sokal andRohlf 1981).

These analyses collectively sought to provide the informationneeded to discriminate single-species populations from those com-posed of interspecific hybrids, multiple species, or obligately asex-ual clones. Populations had to satisfy one of two conditions to berecognized as consisting of a single species: they had to bemonomorphic at all of the allozyme loci examined or, if polymor-phic, they could not show a significant (p < 0.05) GDR deviation.

Diagnosis of species boundariesInitial studies focused on examining the genetic relationships

among populations that were designated as consisting of a singlespecies, based on the above criteria. These analyses were carriedout separately on the collections from South Australia and WesternAustralia to avoid interpretational complications that might derivefrom regional variation in gene frequencies. A UPGMA of Nei’sgenetic distance was employed to examine the patterning of ge-netic divergence among populations within each area. Because datawere unavailable for four loci in several populations, the distancemeasures were based on allelic frequencies at the six loci wherethere were no missing data. Populations showing a genetic distanceless than 0.15 were treated as conspecific, while those showing agenetic distance greater than 0.30 were recognized as distinct spe-cies. The morphological correspondence between each taxon recog-nized through allozyme analysis and reference populations of thefour species known from Australia was subsequently assessed toassign each genetic group to either a known or a new species. Pop-ulations of putative species were later examined for diagnosticallelic substitutions that distinguished them from other groups.

Following determination of diagnostic alleles between each pairof taxa, this information was used to examine the taxonomic com-position of populations that were initially excluded from analysis.Populations showing a heterozygote deficit were examined to see ifthis reflected the presence of two or more species, as indicated bythe co-occurrence of individuals homozygous for the fixed allelicdifferences characteristic of species collected in other habitats.Conversely, populations with a heterozygote excess were examinedto ascertain if they reflected interspecific hybridization, as indi-cated by fixed heterozygosity at loci showing allelic substitutionsbetween species.

Results

Taxonomic diversity in Western AustraliaEleven of the 20Daphniopsisspp. populations from this

region were polymorphic at one or more loci, but 5 of these

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Site Species Latitude (S) Longitude (E)

SA1 T 35°33′ 139°22′SA2 A, T 35°36′ 139°23′SA3 T 35°50′ 139°22′SA4 T 35°55′ 139°29′SA5 T 36°14′ 139°42′SA6 T 36°22′ 139°46′SA7 T 36°27′ 139°47′SA8 T 36°28′ 139°47′SA9 P × T 33°50′ 138°51′SA10 Q 33°55′ 138°53′SA11 T 34°27′ 136°02′SA12 A 34°34′ 135°34′SA13 T 34°18′ 135°36′SA14 T 34°17′ 135°30′SA15 A, T 34°17′ 135°30′SA16 T 34°19′ 135°30′SA17 T 34°20′ 135°29′SA18 A 34°08′ 135°30′SA19 T 34°10′ 135°30′SA20 T 34°09′ 135°32′SA21 T 35°56′ 135°16′SA22 T 33°47′ 135°17′SA23 T 33°46′ 135°06′SA24 T 33°24′ 134°53′SA25 A, Q 34°52′ 139°21′SA26 T 34°54′ 137°40′SA27 T 35°00′ 137°36′SA28 T 37°13′ 139°47′

Table 1. Geographic locations ofDaphniopsisspecies present in35 South Australian (SA) habitats and 20 Western Australian(WA) habitats.

SA29 T 37°16′ 139°48′SA30 T 37°17′ 139°49′SA31 T 37°28′ 140°00′SA32 T 35°55′ 137°39′SA33 A, T 35°56′ 137°34′SA34 T 35°37′ 137°33′SA35 T 35°40′ 137°34′WA1 Q 31°00′ 119°38′WA2 P 29°42′ 115°48′WA3 P 30°06′ 116°38′WA4 P 30°10′ 116°20′WA5 P 32°00′ 115°30′WA6 P 32°00′ 115°30′WA7 P 32°00′ 115°30′WA8 P 30°22′ 117°07′WA9 T 31°12′ 115°47′WA10 Q, W 31°32′ 118°04′WA11 Q 32°57′ 119°34′WA12 Q, W 33°18′ 118°28′WA13 T 29°52′ 114°58′WA14 T 29°53′ 116°01′WA15 T 30°06′ 116°38′WA16 W 33°03′ 115°42′WA17 Q, P 33°25′ 117°22′WA18 Q, P 33°25′ 117°22′WA19 W 33°19′ 117°20′WA20 T 30°38′ 116°00′

Note: Species are designated as follows: A,D. australis; P, D. pusilla;Q, D. queenslandensis; T, D. truncata; W, D. wardi.

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populations showed a significantly lower GDR than expectedin single-species populations (Fig. 2). Analysis of genetic re-lationships among the nine monomorphic populations andthe six populations with a normal GDR indicated the pres-ence of four species represented by two, two, seven, andfour populations respectively (Fig. 3). Morphological com-parisons indicated that the commonest species wasD. pusilla,while one of the other species wasD. queenslandensis. Theother two taxa, one represented by four populations and theother by two, were previously unrecognized and are de-scribed here asD. truncata and D. wardi.

The large genetic distances among the species reflectedthe marked divergence in their allelic arrays. Species weresufficiently differentiated that they could be diagnosed usingonly two loci: Apk and Ldh (Table 2). Populations ofD. wardi and D. queenslandensispossessed uniqueLdh al-leles, while the two species producing one-egged ephippia,D. pusilla and D. truncata, shared a different allele at thislocus. However, the latter pair of species were reliably dis-criminated by their fixation for alternate alleles atApk.

Levels of genetic variability differed among the species.Populations ofD. pusilla were monomorphic at all loci ex-

cept Pgm and Mpi, and no more than two alleles were de-tected at these loci in any population. By contrast, popula-tions of D. truncata were extremely variable and allelicdiversity was so high at some loci, such asPgm and Mpi,that allelic variants could not be reliably discriminated. Al-though fewer populations were available for comparison,D. wardi appeared to possess greater variability thanD. queens-landensisbut less diversity thanD. truncata.

Subsequent investigations focused on characterizing thefive populations excluded from the UPGMA analyses be-cause of their low GDRs. Four of these populations repre-sented cases of species coexistence, as demonstrated by theco-occurrence of individuals showing the diagnostic allelicarrays of two species.Daphniopsis pusillawas the dominantspecies (>95%) at two sites (WA17, WA18), where it co-occurred withD. queenlandensis, while the latter species co-occurred withD. wardi at two other sites (WA10, WA12),with each species dominating one habitat. The allelic arrayof the other population (WA14) suggested that it representeda population of D. truncata, a result confirmed by theUPGMA analysis, which showed its close affinity to theother four populations of this species (Fig. 3).

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Fig. 2. Genotypic characteristics of 11 polymorphic populations ofDaphniopsisspp. from Western Australia. The GDR is the ratio ofobserved genotypic diversity to that expected in a sexual population. Open symbols indicate populations in which the GDR did not dif-fer significantly from that expected and solid symbols represent populations with a significantly lower GDR. LOGP is the negative logprobability that genotypic arrays in a population were in HW equilibrium; a value of 3 indicates that the probability of congruence is10–3. F is the inbreeding coefficient.

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Fig. 3. UPGMA of allozyme divergence among 15 populations ofDaphniopsisspp. from Western Australia whose genotypic diversityapproximated that expected in a single-species population. One population with a low GDR but an allelic array suggesting its single-species status is included (*).

Locus AlleleD. pusilla(n = 7)

D. truncata(n = 4)*

D. wardi(n = 2)

D. queenslandensis(n = 2)

Aat 1.30 — — 1.00 1.001.14 — 0.72 — —1.00 1.00 0.25 — —0.90 — 0.03 — —

Ao 1.25 — — 1.00 1.001.00 1.00 1.00 — —

Apk 1.08 — 1.00 — —1.00 1.00 — 1.00 —0.91 — — — 1.00

Fum 1.15 — 0.01 — —1.00 1.00 0.69 1.00 1.000.88 — 0.30 — —

Gpi 1.17 — — 0.03 —1.10 — — 0.80 —1.00 1.00 0.04 0.12 —0.91 — 0.95 0.05 1.000.81 — 0.01 — —

Ldh 1.04 — 0.02 — —1.00 1.00 0.98 — —0.94 — — — 1.000.70 — — 1.00 —

Me 1.00 1.00 0.01 — —0.88 — 0.99 — —0.83 — — 1.00 1.00

Mdh 1.15 —- 1.00 1.00 1.001.00 1.00 — — —

*n = 2 for Aat.

Table 2. Mean gene frequencies at eight allozyme loci for monospecific populations belongingto four species ofDaphniopsisdetected in Western Australia.

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Taxonomic diversity in South AustraliaAll 35 Daphniopsisspp. populations from this area were

polymorphic at one or more allozyme loci. Analysis ofgenotypic frequencies indicated that six of these populationsshowed a significantly lower GDR than expected in single-species populations (Fig. 4). Cluster analysis of the other 29populations indicated the presence of three genetically diver-gent groups, including 26, 2, and 1 populations, respectively(Fig. 5). Morphological studies indicated that the dominantspecies wasD. truncata, while the rare taxa wereD. austra-lis and D. queenslandensis. These three taxa could bereadily discriminated using allozyme markers (Table 3). Thetwo most morphologically similar species (D. australis,D. queenslandensis) showed allelic substitutions atAo, Apk,and Mdh. Daphniopsis truncatashowed the highest allelicdiversity, which complicated its diagnosis, but possessedunique alleles atLdh and Apk.

Subsequent analysis focused on inspecting genotypic ar-rays in the six populations with deviant GDR values. Four ofthese populations represented cases of species coexistence.In three habitats (SA2, SA15, SA33), individuals possessedallelic arrays typical of eitherD. australis or D. truncata,with the latter species constituting 75–95% of each popula-tion. Daphniopsis australisand D. queenslandensiscoex-isted in one other habitat (SA25), the latter constituting 85%of the individuals. The other two populations did not showindividuals with fixed allelic differences characteristic ofmultispecies assemblages. One of these populations (SA28)

showed weakly significant deviation from the GDR, andlikely represented a single-species population ofD. truncata,a conclusion supported by its close genetic affinity withother populations of this species (Fig. 5). By contrast, the re-maining population (SA9) showed both an extremely lowGDR and strong deviation from HW equilibrium at severalloci, owing to heterozygote excess (Fig. 4). This population,which included three clones showing heterozygosity at sev-eral loci (Apk, Mdh, Me) for alleles that are diagnostic be-tween D. pusilla and D. truncata, was classified as a F1hybrid. The UPGMA confirmed that this population was ge-netically divergent from all the other South Australian popu-lations (Fig. 5).

Species abundances and distributionsFollowing the taxonomic categorization of all populations,

it was possible to ascertain species distributions (Fig. 1, Ta-ble 4). The results indicated that four species ofDaphniopsiswere present in Western Australia, withD. pusilla andD. truncatajointly dominant. The South Australian fauna in-cluded three species, withD. truncataoccurring in 85% ofthe habitats. This species showed the broadest range of sa-linity tolerance, occupying habitats with conductivities rang-ing from 23 100 to 101 000µS/cm. Its tolerance of highsalinity was indicated by the fact that 19 of the 27 SouthAustralian habitats containing only this species had conduc-tivities in excess of 50 000µS/cm. By comparisonD. austra-lis occurred over a narrower salinity range (22 400 – 48 700

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Fig. 4. Genotypic characteristics of 35 populations ofDaphniopsisspp. from South Australia. Open symbols indicate populations inwhich the GDR did not differ significantly from that expected, while solid symbols represent the six populations with a significantlylower GDR. See Fig. 2 for details.

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µS/cm), whileD. queenslandensiswas restricted to habitatswith conductivities ranging from 24 000 to 27 400µS/cm.

Species co-occurred in 15% of habitats, with two speciesof Daphniopsis present in 4 of 20 habitats in WesternAustralia and 4 of 35 habitats in South Australia. Activecoexistence only occurred in habitats with relatively lowconductivities (<40 000µS/cm). However, in some otherhabitats, ephippia of a second species were detected, al-though only one species was active at the time of collection,perhaps because salinities were too high for the other taxon.When analysis was restricted to cases of active co-occurrence,it was apparent that species showed a varied incidence ofcohabitation (Table 4). For example,D. truncataco-occurredwith another species in only 3 of 35 habitats, in each casewith D. australis. By contrastD. queenslandensisco-occurredwith one of three different species in five of the eight habitatswhere it was collected.

Taxonomic diversity in Australian Daphniopsisspp.This study has revealed two undescribed species of

Daphniopsis, necessitating both their formal description anda diagnostic key permitting discrimination of the six speciesof Daphniopsis now known to occur in Australia. Bothallozyme and morphological keys to these taxa follow,togther with taxonomic descriptions forD. truncata andD. wardi.

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Fig. 5. UPGMA of allozyme divergence among 29 populationsof Daphniopsisspp. from South Australia whose genotypic di-versity approximated that expected in a single-species population.One population with a low GDR but an allelic array suggestingits single-species status (*) and one population showing evidenceof a hybrid origin (**) are included. Locus Rf

D. truncata(n = 25)*

D. australis(n = 2)

D. queenslandensis(n = 1)

Aat 1.07 0.36 0.12 —1.00 0.57 0.88 0.360.94 0.07 — —0.82 — — 0.64

Ao 1.32 — 1.00 —1.10 0.01 — 1.001.00 0.67 — —0.95 0.32 — —

Apk 1.08 1.00 — —1.00 — 1.00 —0.91 — — 1.00

Fum 1.08 0.01 0.12 1.001.00 0.97 0.88 —0.84 0.02 — —

Gpi 1.04 0.11 — —1.00 0.27 — —0.95 0.05 — —0.91 0.52 0.68 1.000.82 0.04 0.32 —0.80 0.01 — —

Ldh 1.12 0.02 — —1.03 — — 0.271.00 0.98 — —0.91 — — 0.730.70 — 1.00 —

Mdh 1.15 0.66 — 1.001.00 0.34 1.00 —

Me 0.88 0.99 0.80 1.000.77 0.01 0.20 —

Mpi 1.12 0.01 — —1.08 0.02 — —1.05 0.15 — —1.00 0.57 0.22 —0.90 0.11 0.35 —0.86 0.11 0.43 1.000.74 0.02 — —0.72 0.01 — —

Pgm 1.07 0.01 — 0.191.04 0.13 — 0.691.00 0.70 — 0.120.93 0.15 1.00 —0.87 0.01 — —

*n = 7, 24, 24, 22, 14, and 23 forAat, Fum, Ldh, Mdh, Mpi, andPgm,respectively.

Table 3. Mean gene frequencies at 10 allozyme loci formonospecific populations of three species ofDaphniopsisdetected in South Australia.

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Allozyme key

All allelic mobilities employed in this key are relative to the dominant allele inD. pusilla from Rottnest Island, with allozymesanalyzed on cellulose acetate gels in a Tris glycine (pH 8.5) buffer.

1a. Apkwith faster mobility (Rf = 1.08) than the standard.................................................................................................D. truncata1b. Apkwith the same mobility as the standard or slower ..................................................................................................................2

2a. Apkwith slower mobility (Rf = 0.91) than the standard ..................................................................................D. queenslandensis2b. Apkwith the same mobility as the standard...................................................................................................................................3

3a. Ldhwith slower mobility (Rf = 0.70) than the standard................................................................................................................43b. Ldh with the same mobility as the standard or faster ....................................................................................................................5

4a. Mdh with faster mobility (Rf = 1.15) than the standard ....................................................................................................D. wardi4b. Mdh with the same mobility as the standard ................................................................................................................D. australis

5a. Ao with faster mobility (Rf = 1.24) than the standard ...........................................................................................D. quadrangula5b. Ao with the same mobility as the standard ......................................................................................................................D. pusilla

Morphological keyMorphological differences among parthenogenetic fe-

males are often so subtle that identifications are problematic.By contrast, variation in ephippial morphology among spe-cies is dramatic, providing reliable characters for speciesrecognition. This reliance on an identification system based

on the ephippial stage is not a serious constraint becauseephippial females are present in most samples. Moreover, byrelying on traits that are apparent in ephippia, it is possibleto determine the complete species assemblage in habitatswhere only a fraction of the taxa are active in the plankton atthe time of collection.

Key to Australian Daphniopsisspp. (ephippial females)

1a. Ephippial case with one egg chamber............................................................................................................................................21b. Ephippial case with two egg chambers ..........................................................................................................................................3

2a. Dorsal surface of the ephippium lacking spinescence (Fig. 9C), female rostrum bluntly rounded (Fig. 9A) ...........D. truncata2b. Dorsal surface of ephippium with spinescence (Fig. 9C), female rostrum angular (Fig. 9A).......................................D. pusilla

3a. Ephippium with elongate anterior and posterior processes (Fig. 7b) ...................................................................D. quadrangula3b. Ephippium lacking an elongate posterior process (Figs. 7d, 7f, 7h) .............................................................................................4

4a. Ephippium with an elongate anterior process (Figs. 7d, 7f) .........................................................................................................54b. Ephippium lacking an elongate anterior process (Fig. 7h) ..........................................................................................D. australis

5a. Posterior process of ephippium very reduced (Fig. 7f); dorsal surface of ephippial female smoothly rounded (Fig. 7e)..................................................................................................................................................................................D. queenslandensis5b. Posterior process of ephippium distinct (Fig. 7d); dorsal surface of ephippial female angular (Fig. 7c).......................D. wardi

Species descriptions

Daphniopsis wardisp.nov. (Fig. 6)ETYMOLOGY: This species is named in honour of Dr. Robert

D. Ward, who played a critical role in the launch of geneticstudies on natural populations of cladoceran crustaceans.

TYPE LOCALITY: Lake Preston, Western Australia (WA16), alarge coastal salt-water lake, approximately 120 km south ofPerth on the boundary of Yalgorup National Park (33°03′S,115°42′E). This site was collected on October 24, 1995.

HOLOTYPE: An ephippial female in ethanol deposited in theAustralian Museum. Accession No. P57752.

ALLOTYPE: A mature male in ethanol deposited in the Aus-tralian Museum. Accession No. P57753.

PARATYPES: Four parthenogenetic females, 10 ephippial fe-males, and 10 ephippia, preserved in ethanol and depositedin the Australian Museum. Accession No. P57754.

OTHER MATERIAL EXAMINED: Western Australia: WA10: sa-line pond on right when heading east, 2.5 km before HinesHill and 22.1 km west of Merredin; WA12: saline pond on

WesternAustralia

SouthAustralia

D. australis — 2D. wardi 2 —D. pusilla 7 —D. quadrangula — —D. queenslandensis 2 1D. truncata 5 27F1 (D. truncata× D. pusilla) — 1D. australis+ D. truncata — 3D. australis+ D. queenslandensis — 1D. wardi + D. queenslandensis 2 —D. pusilla + D. queenslandensis 2 —

Total 20 35

Table 4. Daphniopsisspecies assemblages from 20 habitats inWestern Australia and 35 saline habitats in South Australia.

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Fig. 6. Daphniopsis wardiin lateral view. (a–f) Female, showing the body (a and b), head (c and d), abdomen (e), postabdomen (f),ephippium (g), and dorsal surface (h). (i–k) Male, showing the body (i), flagellum (j), and abdomen (k). The specimens ina and cwere collected in Tammin, Western Australia, on October 2, 1995, and the remaining specimens were collected from Lake Preston,Western Australia, on October 24, 1995.

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right when heading south from Lake Grace to Albany,26.4 km south of Lake Grace; WA19: saline pond on bothsides of the road, 3.7 km east of Wagin on road to Dumble-yung.

HABITAT: This species appears to be restricted to saline pondsand lakes.

DISTRIBUTION: This species is apparently endemic to WesternAustralia.

BREEDING SYSTEM: Allozyme analysis indicates thatD. wardireproduces by cyclic parthenogenesis.

HYBRIDIZATION : This species has been found to co-occurwith D. queenslandensisbut no hybrids were detected.

NARRATIVE: Daphniopsis wardiis an uncommon memberof the saline lake fauna in Western Australia.

FEMALE: Body (Figs. 6a, 6b): The body length of ovigerousfemales ranges from 1.9 to 2.1 mm. A tail spine is absentfrom both juvenile and adult females. The eternal spinescenceon the dorsal and ventral carapace margins is weak. However,the internal margin of the ventral carapace has elongate,plumose setae anteriorly and prominent setae posteriorly.The carapace is intensely melanized.Head (Figs. 6c, 6d):The compound eye and ocellus are large and darkly melanized,but the optic nerve lacks melanin granules. The antennularmounds are very distinct, stalk-like projections.Abdomen(Figs. 6e, 6f): All four abdominal processes are weakly de-veloped. The postabdominal surface is smoothly roundedand possesses 12–14 anal spines.Ephippium(Figs. 6g, 6h):The ephippium has two egg chambers and both an anteriorand a posterior process. The anterior process, which hasprominent spines along its ventral margin, is elongate andtypically splits along its medial line. The posterior process ismuch shorter, but distinct. The dorsal ridge of the ephippiumhas a rough appearance, reflecting the presence of shortspines.

MALE: Figs. 6i–6k. The body length of the mature maleranges from 1.2 to 1.6 mm. There is no tail spine and the ex-ternal spinescence on the carapace is inconspicuous. Theflagellum of mature males is very elongate, nearly 5 times aslong as the aesthetascs. The postabdomen, which has a smoothlyrounded surface, has conspicuous anal spines.

DIFFERENTIAL DIAGNOSIS: The four species ofDaphniopsisthat produce two-egged ephippia can be readily distinguished(Fig. 7). In ephippial females ofD. queenslandensisthe dorsalcarapace margin isrounded (Fig. 7e), while in the otherspecies it is angular, giving them a distinctly “hump-backed”appearance (Figs. 7a, 7e, 7g). The body shapes of femaleD. australisandD. wardi are similar, with a concave posteriorangle as opposed to the convex angle ofD. quadrangula.The ephippia themselves can be readily discriminated: thoseof D. quadrangulahave an elongate posterior process (Fig. 7b),while those of the other species (Figs. 7d, 7f, 7h) have ashort posterior process. The ephippia ofD. australis andD. queenslandensiscan be differentiated by the presence ofa prominent anterior process inD. queenslandensisversusits lack in D. australis.

Daphniopsis truncatasp.nov. (Fig. 8)ETYMOLOGY: The specific name jointly derives from the

truncated rostrum and the short second antennae that charac-terize this species.

TYPE LOCALITY: Robe Lake (SA28), a coastal saline lake, onthe right side of Nora Creina Tourist Drive, 6.0 km south of

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Robe, South Australia (37.13°S, 139.47°E). This site wascollected on November 24, 1996.

HOLOTYPE: An ephippial female in ethanol deposited in theAustralian Museum. Accession No. P57755.

ALLOTYPE: A mature male in ethanol deposited in the Aus-tralian Museum. Accession No. P57756.

PARATYPES: Ten parthenogenetic females, 1 ephippial fe-male, and 10 males in ethanol deposited in the AustralianMuseum. Accession No. P57757.

OTHER MATERIAL EXAMINED: Western Australia: WA9: Lakeon left side of dirt road, 3 km west of its intersection withHighway 1, approximately 45 km north of Muchea and 6 kmnorth of Lake Beermullah; WA13: saline pond on left,1.6 km north of Coolimba on road to Illawong; WA14:saline pond on right, 82 km north of Moora betweenMarchagee and Coorow; WA15: saline pond on right,14.7 km west of Wubin when heading towards Gunyidi;WA20: saline pond on right, 4.1 km west of Moora on roadto Dander.South Australia: Populations ofD. truncatawereexamined from 30 South Australian habitats, all in closeproximity to the coast (Fig. 1, Table 1). Twenty-six popula-tions occurred in unnamed ponds or lakes, while four sites,including the type locality, were named. They includedSA23, Lake Tungetta, approximately 30 km south of Ellis-ton; SA31, Wooley Lake, 1.4 km north of Beachport; SA33,Tea Tree Lagoon, on the road to Cape Lirois, Kangaroo Is-land.Tasmania: Tas9: Bar Lagoon, a saline lake, situated ap-proximately 15 km west of Ross (42.03°S, 147.20°E).

HABITAT: This species occurs in saline lakes and ponds inSouth Australia, Tasmania, and Western Australia. Popula-tions from South Australia showed a broad salinity toler-ance, occurring in habitats with conductivities ranging from23 100 – 101 000µS/cm.

HYBRIDIZATION : A population of D. pusilla × D. truncatahybrids was detected in Porter’s Lake, South Australia(SA9).

NARRATIVE: Daphniopsis truncatais apparently the mostabundantDaphniopsisspecies in Australia. It was previouslyconfused withD. pusilla, but several morphological charac-ters permit the reliable discrimination of these two species.

FEMALE: Body (Figs. 8a, 8b): The body lengths of ovigerousfemales range from 1.4 to 2.9 mm. A tail spine is absentfrom both juvenile and adult females. The external spinescenceon the dorsal and ventral margins of the carapace is weak.However, the internal margin of the ventral carapace haselongate, plumose setae anteriorly and pronounced setaeposteriorly. The carapace is normally melanized.Head(Figs. 8c, 8d): The compound eye and ocellus are large anddarkly melanized, and the optic nerve itself often possessesmelanin granules. The antennular mounds are conspicuousbut lack the stalk-like projections apparent in some otherspecies ofDaphniopsis. Abdomen(Figs. 8e, 8f): All four

abdominal processes are weakly developed. The postabdominalsurface is smoothly rounded and has 12–15 anal spines.Ephippium(Figs. 8g, 8h): The ephippium has just a singleegg chamber and lacks both an anterior and a posterior pro-cess. Because it lacks pubescence, the dorsal ridge of theephippium appears smooth and shiny.

MALE: Figs. 8i–8k. The body length of the mature maleranges from 1.2 to 1.7 mm. There is no tail spine and the ex-ternal spinescence on the carapace is very weak. The flagellumof mature males is prominent, reaching 5 times the length ofthe aesthetascs. The postabdominal surface is distinctly sinuateand possesses conspicuous anal spines.

DIFFERENTIAL DIAGNOSIS: The two species ofDaphniopsisthat produce one-egged ephippia can be distinguished usinga variety of characters (Fig. 9). Adult femaleD. truncatapossess a bluntly rounded rostrum, while those ofD. pusillahave an angular rostrum (Fig. 9A). Pre-ephippial femaleD. truncatatypically show a deeper indentation of the dorsalcarapace margin than those ofD. pusilla (Fig. 9B). As well,the ephippial ridge ofD. truncata is smooth, while that ofD. pusilla is densely pubescent (Fig. 9C).

Discussion

While the saline waters of other continents contain onlyone or two species ofDaphniopsis, diversity is considerablyhigher in Australia. Based upon their study of variation inephippial morphology, Kokkinn and Williams (1987) sug-gested that there were at least six species on the continent.Morphological studies subsequently led to the description ofspecies producing four of these six ephippial types (Sergeev1990a, 1990b), but the parental taxa of the other twoephippial variants remain unknown.Daphniopsis truncataproduces an ephippium similar to that of variant 1 reportedby Kokkinn and Williams (1987), filling one of these gaps.However, the other new species,D. wardi, possesses a novelephippial morphology, suggesting that at least one more spe-cies awaits discovery, so the Australian fauna includes atleast seven species.

Aside from clarifying the taxonomic diversity of Austra-lian Daphniopsis, the present study has provided some infor-mation on their genetic attributes. The allozyme resultsindicate that each of the six species possesses diagnosticgene substitutions allowing its discrimination from the othertaxa. The extent of this genetic divergence suggests thatthese species do not share a recent ancestry and this conclu-sion has been confirmed through studies of DNA sequencedivergence (Hebert et al.).3 Allozyme variation in all specieswas sufficient for verifying that their populations normallyreproduce by cyclic parthenogenesis. However, the degree ofvariation appeared to differ among taxa. For example, thewidely distributed speciesD. truncata showed far more

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Fig. 8. Daphniopsis truncatain lateral view. (a–f) Female, showing the body (a and b), head (c and d), abdomen (e), postabdomen (f),ephippium (g), and dorsal surface (h). (i–k) Male, showing the body (i), flagellum (j), and abdomen (k). The specimens ina, c, g, andh are from site SA24, those inb, i, j, andk are from WA13, and those ind, e, and f are from WA14.

3P.D.N. Hebert, E.A. Remigio, J.K. Colbourne, D.J. Taylor, and C.C. Wilson. Accelerated molecular evolution in halophilic crustaceans.Submitted for publication.

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allozyme diversity than the allied, but more narrowly distrib-uted, D. pusilla. Interestingly, a population ofD. truncataexamined from Tasmania showed considerable variation(personal observation) despite the presence of fewer than 20saline habitats on this island.

The present study has extended understanding of the dis-tributional patterns ofDaphniopsisspecies in Australia. Twospecies,D. australis and D. quadrangula, appear to be en-demic to the southeast, while two other species,D. wardiand D. pusilla, are restricted to the west.Daphniopsis

queenslandensisseems to have the broadest distribution, oc-curring over at least the southern half of the continent, whileD. truncata is the commonest species, because of its preva-lence in areas of South Australia and Western Australia thatare rich in saline habitats. The local distributional pattern ofeach species is likely strongly influenced by its tolerance ofsalinity. CertainlyD. truncatashowed much higher salinitytolerance than the other species in South Australia. Variationin UV resistance may also be important, asD. queens-landensis, which had weaker pigmentation than the other

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Fig. 9. Diagnostic morphological differences betweenD. truncataand D. pusilla. (A) Female head (left) and female rostrum (right).(B) Pre-ephippial female. (C) Dorsal surface of ephippium.

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taxa, tended to occur in more turbid habitats (personal obser-vation). In habitats where it co-occurred with other species,it was present in deeper water, while melanized speciesdominated the shallows, a distributional pattern similar tothat noted for melanized and unmelanized clones in theDaphnia pulexcomplex (Hebert and Emery 1990).

As co-occurrence was detected in approximately 15% ofhabitats, and species often synchronously engaged in sexualreproduction (personal observation), there were opportuni-ties for interspecific hybridization. Yet hybrids were detectedin only a single habitat, Porter’s Lake, South Australia,where the population consisted of three F1 hybrid cloneswhose allozyme phenotypes suggested their derivation froma cross betweenD. pusilla andD. truncata. The detection ofhybrids in this inland lake was significant, as all 30 SouthAustralian populations ofD. truncata were restricted tocoastal areas, whileD. pusilla is so far unknown from thisarea. This case of hybridization was unusual, as cladoceranhybrids are normally restricted to regions where their paren-tal taxa co-occur (Hebert and Wilson 1994). However, sincethese hybrids reproduced by obligate parthenogenesis, abreeding-system transition detected in other cases of clado-ceran hybridization (Hebert 1987; Little et al. 1997), theymay have originated in another area. The rarity of hybridsbetween Australian species ofDaphniopsisprovides furtherevidence for their genetic divergence, as past work has in-dicated that reproductive isolation evolves slowly in clado-cerans. For example, hybridization is common among speciesof Daphnia in North America that diverged within the last10 million years (Colbourne and Hebert 1996).

The results of this study support the occurrence of a radia-tion of Daphniopsisin the saline lakes of Australia that isunmatched in similar habitats on any other continent. ThisDaphniopsisdiversity parallels that noted in several othercrustacean genera from Australian saline lakes, includingthe anostracan genusParartemia (Geddes 1981) and theostracod genusMytilocypris (DeDeckker 1981). The lack ofsimilar radiations on other continents suggests that salinehabitats have been abundant in Australia for a substantial pe-riod of time. This conclusion is supported by the markedallozyme divergence among species ofDaphniopsis, sug-gesting that the lineages are not the result of a recent burstof divergence. Future studies examining the extent of DNAsequence divergence among the component species of gen-era restricted to saline environments should provide a bettersense of the origins of the diversity that uniquely character-izes the saline lakes of Australia.

Acknowledgements

Funding for this project was provided by a research grantfrom the Natural Sciences and Engineering Research Coun-cil of Canada (NSERC) to P.D.N.H. C.C.W. was supportedby NSERC and Australian Research Council postdoctoralfellowships. We thank Bill Williams, Jennie Chaplin, andBob Ward for access to laboratory space in Adelaide, Perth,and Hobart, respectively. Young Um provided assistancewith data analysis, while Teri Crease provided helpful com-ments on an earlier draft of the manuscript.

References

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