CSIRO PUBLISHING www.publish.csiro.au/journals/ajz Australian Journal of Zoology, 2009, 57,77–84

Co-occurrence of two tadpole shrimp, cf. australiensis ( : ), lineages in middle Paroo, north-western New South Wales, with the first record of Triops hermaphrodites for the Australian continent

Gopal Murugan A, Hortencia Obregón-BarbozaA, Alejandro M. Maeda-Martínez A,C and Brian V. Timms B

ACentro de Investigaciones Biológicas del Noroeste, Mar Bermejo 195, La Paz, Baja California Sur, C. P. 23090, México. BSchool of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW 2308, Australia. CCorresponding author. Email: [email protected]

Abstract. The only species of the Triops in Australia, T. australiensis, is found to reproduce by gonochorism. Morphological and reproductive data and molecular analyses of fragments of mitochondrial genes 12S rRNA (12S) and cytochrome oxidase I (COI) indicate that a Triops population from the middle Paroo in north-western New South Wales is composed of two different entities, Triops cf. australiensis lineage A, and T. cf. australiensis lineage B. Gonad histology in individuals with ovisacs of lineage A revealed no evidence of testicular tissue; however, large testicular lobes were found in individuals with ovisacs of lineage B, indicating that they were anatomically hermaphrodites. This is the first record of Australian hermaphroditic Triops. For eachlineage, a single haplotype of each gene was obtained. Molecular genetic distance and phylogenetic analyses confirmed the closer relationship and monophyly of the two lineages with T. australiensis (GenBank) when compared with Triops species from other continents. COI haplotypes of lineage A and lineage B differed by 7.7% from each other and differed by 10.2% and 9.6% from a published T. australiensis sequence, respectively. The 12S haplotypes of lineage A and lineage B differed by 3.3% from each other and differed by 2% and 2.5% from a published T. australiensis sequence, respectively. Our results suggest that the two co-occurring Triops lineages probably represent two species that are distinct from T. australiensis.

Introduction (Pallas, 1793) (cited as glacialis) from Greenland and Triops australiensis (Spencer & Hall, 1895) and Lepidurus apus Spitzbergen, and Lepidurus apus (cited as Lepidurus productus) (Linnaeus, 1758) are the only recognised taxa of tadpole shrimp and Triops cancriformis (Bosc, 1801) (cited as Apus occurring in Australia (Longhurst 1955a; Williams 1968; Fryer cancriformis), both from central Europe. Since then, different 1988; Williams and Busby 1991). T. australiensis is also known morphs of tadpole shrimp exhibiting hermaphroditic or from Madagascar as T. australiensis sakalavus (Nobili, 1905), gonochoric reproduction have been reported for the same species while L. apus is a widely distributed species with records in (Longhurst 1955b; Grigarick et al. 1961; Akita 1971; Scanabissi Europe, North Africa, Asia, the Americas, and and Mondini 2002). Gonads of individuals with ovisacs from (Longhurst 1955a), although this has been debated and several male-less populations of the European Triops have been studied species have been resurrected (Lynch 1972; Rogers 2001). Fryer histologically to elucidate the mode of reproduction (Zaffagnini (1988) suggested that the Australian Lepidurus may be a different and Trentini 1980). Trentini and Scanabissi (1982) found, in species, and that the widely distributed taxa in the Notostraca T. cancriformis from , that the sex ratio was heavily biased proposed by Linder (1952) and Longhurst (1955a) may consist of in favour of individuals with ovisacs with a rudimentary form many races or several cryptic species. Genetic and morphological of hermaphroditism, and concluded that these populations evidence supporting the latter possibility has increased in reproduce parthenogenetically. Sassaman (1991), on the basis of recent years (Sassaman et al. 1997; King and Hanner 1998; sex ratio and pedigree studies, proposed that those Triops Rogers 2001; Murugan et al. 2002; Korn and Hundsdoerfer populations from North America that were composed mostly of 2006; Korn et al. 2006; Zierold et al. 2007). individuals with ovisacs (putative hermaphrodites) practise Over a century ago, Bernard (1891, 1895) reported the androdioecious reproduction. Androdioecious populations presence of spermatic cells within ovarian tissue in individuals consist of small proportions of males, and of amphigenic and with ovisacs of three tadpole shrimp, Lepidurus arcticus monogenic hermaphrodites that are able to produce offspring by

CSIRO 2009 10.1071/ZO08084 0004-959X/09/020077 78 Australian Journal of Zoology G. Murugan et al. selfing or by mating with a male (Sassaman 1989, 1995; Davidson’s solution (Bell and Lightner 1988). Serial sections Sassaman and Weeks 1993). (3–6 mm) were obtained and stained with hematoxylin and eosin Longhurst (1955a) reported Lepidurus apus as a species dyes. For corroboration of testicular tissue, sections were stained showing both hermaphroditic and gonochoric (cited as bisexual) with Fulgen picromethyl blue (Howard and Smith 1983). reproduction; however, no males have been reported for this Analyses of fragments of the mitochondrial cytochrome species from Australia. Although the sex ratio of the original oxidase subunit I (COI) and 12S rRNA (12S) genes were carried collection of T. australiensis was strongly biased towards out on 14 adult specimens preserved in 100% ethanol after individuals with ovisacs (i.e. 6 males and 52 individuals with recordingtheirmorphologicalcharacteristics.TotalDNA(tDNA) ovisacs), Spencer and Hall (1896) reported no histological was obtained from abdominal muscle using the Puregene kit evidence of spermatic cells or testicular lobes in ovaries of (Gentra Systems, Inc., D5500A). A fragment of COI was individuals with ovisacs. Since then, T. australiensis has been amplified using primers LCO1490 and HCO2918 (Folmer et al. considered as a gonochoric (cited as bisexual) species (Longhurst 1994). Each 50-mL PCR mixture for COI consisted of tDNA, 1955a). 5 mL10 PCR buffer (200 mM TRIS-HCl, pH 8.4; 500 mM As a part of a broader investigation on the Australian tadpole KCl), 1.5 mM MgCl2, 0.7 mL10mM dNTP, 60 pMol of each shrimp Triops, we report the co-occurrence of two lineages of this primers, and 1 unit of Taq DNA polymerase. Cycling program genus in a single pool from the middle Paroo, New South Wales. was: one cycle at 95C for 5 min; five cycles each of 1 min at The distinction of these lineages is based on morphological and 94C, 1.5 min at 45C, and 1.5 min at 72C; 35 cycles each of molecular (mitochondrial DNA) data, and on gonad histology, 30 s at 94C, 45 s at 45C, and 1.5 min at 72C; and a final from which Triops hermaphrodites were detected. Molecular extension at 72C for 10 min. Sample preparation and cycling genetic distance and phylogenetic analyses confirmed the conditions for 12S consisted of one cycle at 95C for 5 min; closer relationship and monophyly of the two lineages with 40 cycles each of 30 s at 94C, 30 s at 50C, and 1 min at 72C; T. australiensis (GenBank data) when compared with Triops and a final extension for 5 min at 72C (Murugan et al. 2002). species from other continents. Amplified products were purified using Amersham GFX PCR DNA and gel band purification kit (27-9602-01) and sequences of one direction were obtained for both genes. Sequences of Material and methods COI and 12S were aligned using Eye Ball Sequence Editor The population studied came from the Marsilea pool on (ESEE) (Cabot 1998) and Clustal X (Thompson et al. 1997). Bloodwood Station, 130 km north-west of Bourke in the middle The presence of nuclear copies of COI and 12S in the present Paroo catchment, north-western New South Wales, Australia, study is unlikely given the concordant results of two mtDNA with coordinates of 29330S, 144520E (see Timms and Sanders markers that were independently amplified and also the high 2002). Dry soil from the pool was used as a source of Triops similitude of T. cancriformis and T. longicaudatus sequences of resting eggs. The soil was placed in a 600-L fibreglass tank, and this study with the sequences of mitochondrial genome of their Triops were obtained using potable water with total dissolved respective taxon. solids (TDS) = 0.05 g L1 as the culture medium. During the Genetic distances of the sequences of Triops from the Marsilea 23-day culture period, the water temperature ranged from 16 to pool and those from the GenBank database (T. australiensis (COI: 25C, and TDS increased from 0.07 to 0.63 g L1. EF189677; 12S: AY050646), T. cancriformis (COI: GQ144445; The presence of a hemispherical ovisac (oostegopod) in each 12S: AY115603 (); COI and 12S: AB084514 (Japan)), of the 11th thoracopods of the adult tadpole shrimp is the main T. longicaudatus (LeConte, 1846) (COI: GQ144444; 12S: character used to separate females and hermaphrodites from AY115600 (Mexico; COI and 12S: AY639934 (Triops World males (individuals without ovisacs). Thus, a total of 37 Triops Kit, USA), T. granarius (Lucas, 1864) (COI: GQ144446; 12S: specimens were obtained, 30 individuals with ovisacs and seven AY115602 (Japan)) were calculated using maximum composite males. The specimens were deposited in the collections of the likelihood model in Molecular Evolutionary Genetic Analysis Australian Museum, and Centro de Investigaciones Biológicas (MEGA 4) (Tamura et al. 2007). Phylogenetic relationships were del Noroeste, La Paz, México. Using a vernier caliper and a analysed for both genes combined, after a partition homogeneity binocular stereomicroscope the carapace length (CL) was test (Farris et al. 1995), using the maximum parsimony (MP), measured from the anterior edge to the posterior edge on the neighbour joining (NJ), and maximum likelihood (ML) methods middle line of the carapace. Also, we recorded the following in PAUP* 4.0b10 (Swofford 2003) and Bayesian inference (BI) character states (Linder 1952; Longhurst 1955a; Lynch 1972; method in MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003). NJ Rogers 2001; Obregón-Barboza 2007): presence or absence of and ML (heuristic searches, 100 random stepwise additions, second maxilla, colour and spines of dorsal side of carapace, tree–bisection–reconnection (TBR) branch swapping, and spines on carina, position of the nuchal organ anterior margin multiple trees (MulTrees) option) analyses were performed with with respect to the posterior border of the interocular zone of parameters of the nucleotide-substitution model general time the compound eyes, number of spines on sulcus, spines on reversible (GTR) + invariable sites (I) + gamma distribution (G) dorsal-posterior border of telson, total number of body rings, and (with likelihood parameters Base = (0.2908 0.1675 0.1838) number of legless rings. Nst = 6 Rmat = (0.0145 10.0781 5.4819 1.0873 18.6444) A histological analysis of the gonads using a light microscope Rates = gamma Shape = 0.8642 Pinvar = 0.4211) under Akaike was carried out to search for testicular tissue in individuals with Information Criterion (AIC) from ModelTest 3.7 (Posada and ovisacs (Scanabissi and Trentini 1979; Zaffagnini and Trentini Crandall 1998). MP was done using heuristic search settings with 1980). We fixed seven individuals with ovisacs and one male in 2000 stepwise random additions, TBR branch swapping, Co-occurrence of two Triops cf. australiensis lineages Australian Journal of Zoology 79

MulTrees by weighting all characters equally. Statistical support in males consisted of two testes located parallel to the digestive for the reliability of individual branches in phylogenetic trees was tract in the hemocoel (Fig. 2C). We infer the mode of assessed by the use of pseudoreplicates (Felsenstein 1985): 5000 reproduction of this entity is gonochorism, based on the in MP, 10 000 in NJ, and 2000 in ML. BI analysis was run for two histological and sex ratio data. million generations with the same settings of the nucleotide- substitution model applied in the ML and NJ searches. Trees were Molecular data sampled every 100 generations. The first 25% of the trees COI sequences: four sequences (from two females and two males) containing the burn-in phase were discarded. Sequences of three of 664 base-pairs (bp) were obtained representing a single more branchiopod taxa, Lepidurus lemmoni Holmes, 1894 haplotype (GenBank accession number: DQ343234). 12S (Notostraca) (COI: GQ144447; 12S: AY115604), Leptestheria sequences: four sequences (from two females and two males) of dahalacensis (Rüppel, 1837) (Spinicaudata) (COI: EF189670; 560 bp were obtained representing a single haplotype (GenBank 12S: AF494476), and Cyclestheria hislopi (Baird, 1859) accession number: DQ343232). (Cyclestherida), (COI: DQ310631; 12S: AF494478), were included in the phylogenetic analyses. Triops cf. australiensis lineage B Males and individuals with ovisacs that were not molecularly (Fig. 1G–I) analysed were assessed morphologically to one lineage, based on the findings of Obregón-Barboza (2007). She analysed 11 North Material examined American Triops lineages (androdioic and gonochoric) using Twenty-two individuals with ovisacs (CL = 10.5–11.4 mm). Four were used mitochondrial DNA and found that males of one lineage can be for histology and 10 for DNA analysis. distinguished from males of other lineages on the basis of morphological characteristics that they share with individuals Morphological characteristics with ovisacs within the same lineage (mainly the number of spines Second maxilla absent; olive green carapace, with minute spines in the sulcus, the position of the nuchal organ, and the type dispersed on the dorsal surface; carina smooth; anterior margin of (smooth or spinose) of the posterior border of the telson). nuchal organ crosses the posterior border of the interocular zone (Fig. 1G); dorsal-posterior border of telson smooth; sulcus armed Results with 25–30 spines (Fig. 1I); total body rings 35–38; legless body – The Triops population from the Marsilea pool is composed of rings 8 9. two different entities: T. cf. australiensis lineage A and Gonad histology T. cf. australiensis lineage B. They are characterised as follows. The reproductive organs of the individuals with ovisacs consisted Triops cf. australiensis lineage A of two longitudinal gonads located parallel to the digestive tract in the hemocoel. Multibranched tubules with somatic and germ cells (Fig. 1A–F) showing follicles and vitellogenic oocytes were observed along Material examined the gonads (Fig. 2A). Additionally, large testicular lobes were found within the ovary tissue (Fig. 2B). We infer that the mode of – Eight individuals with ovisacs (CL = 13.6 15.3 mm) and seven males reproduction of this entity is hermaphroditism. (CL = 13.8–15.7 mm). Three individuals with ovisacs and one male were used for histology. For DNA analysis, two individuals with ovisacs and two males Molecular data were examined. COI sequences: 10 sequences from 10 individuals with ovisacs Morphological characteristics provided a 664-bp sequence that represented a single haplotype (GenBank accession number: DQ343235). 12S sequences: 10 Second maxilla absent; carapace yellowish with green spots sequences from 10 individuals with ovisacs provided a 577-bp (Fig. 1E), minute spines on the dorsal surface, carina smooth or sequence that represented a single haplotype (GenBank accession spinose; anterior margin of nuchal organ lies just in the posterior number: DQ343233). border of the interocular zone (Fig. 1A, D), dorsal-posterior – border of telson spinose; sulcus armed with 46 54 spines in Genetic distances and phylogenetic analyses individuals with ovisacs and 49–53 in males (Fig. 1C, F); total body rings 35–36 in individuals with ovisacs and 36–37 in males; Sequences of COI obtained in our study represent ~43% of the legless body rings 8–9 in individuals with ovisacs and 10–11 in gene length in the Notostraca. The COI haplotypes of Triops cf. males. australiensis lineage A [haplotype A] and T. cf. australiensis lineage B [haplotype B] differed from each other by 7.7%, and from the T. australiensis sequence (GenBank) by 10.2% and Gonad histology 9.6%, respectively. Of the 51 substitutions in the COI fragment Individuals with ovisacs and males had the typical gonad anatomy between the two haplotyptes, 78% of them were a transitional for gonochoric notostracans. The gonads in individuals with type. Six substitutions were found in the first codon position and ovisacs consisted of two longitudinal ovaries located parallel to the rest were found in the third codon position. With the the digestive tract in the hemocoel, with multibranched tubules T. australiensis sequence, these two haplotypes (A and B) showed of somatic and germ cells showing follicles and vitellogenic 62–67% transistional substitution and 75–79% of the third codon oocytes. No evidence of testicular tissue was found. The gonads positions varied between the two haplotypes and T. australiensis. 80 Australian Journal of Zoology G. Murugan et al.

Fig. 1. (A–C): Male of Triops cf. australiensis lineage A. (D–F): Female of Triops cf. australiensis lineage A. (G–I): Hermaphrodite of Triops cf. australiensis lineage B. (A, D, G): Dorsal view of anterior part of carapace. (B, E, H): Dorsal view of complete carapace. (C, F, I): Dorsal view of sulcus of the posterior part of carapace. ce = compound eye; no = nuchal organ; s = spines on sulcus.

The two 12S haplotypes confirmed the presence of two The genetic distances determined with MEGA software lineages in the Marsilea pool. They showed a difference of 3.4%. with the maximum composite likelihood model are shown in Of 19 base pairs that were different, 15 were transitions and four Table 1. Among the Australian Triops, the genetic distance were transversions. We obtained 560 bp for haplotype A and ranged from 0.021 to 0.034 in 12S and 0.085 to 0.114 in 577 bp for haplotype B, whereas the GenBank sequence of COI. Compared with other Triops species, the Australian T. australiensis contains 506 bp. This sequence needed an Triops showed low genetic divergence with T. longicaudatus. unusual number of insertions to align with other Triops Within congeneric species, genetic distances of 0.008 in 12S sequences. Therefore, we used 395 bp of this sequence in our and 0.024 in COI were observed between T. longicaudatus analysis. In this portion of the 12S fragment, haplotypes A and B from Mexico and the USA (‘Triops World’ Kit) and 0.013 in differed by 3.3% and with T. australiensis (GenBank) by 2.0 and 12S and 0.021 in COI between T. cancriformis from Japan and 2.5%, respectively. Austria (Table 1). Co-occurrence of two Triops cf. australiensis lineages Australian Journal of Zoology 81

The partition homogeneity test for the combined 12S and COI dataset indicated that there was no significant incongruence between sequences of both genes (P = 0.453). The dataset (12S–COI) used in the phylogenetic analyses contained 1044 characters, of which 270 were variable among the Triops sequences. More than 70% of the variable sites were parsimoniously informative. The topology of the consensus tree obtained in MP, NJ, ML and BI was identical and showed that Triops sequences from Australia are monophyletic (Fig. 3). Most of the clades in the phylogenetic trees were supported by bootstrap values higher than 60%. The monophyly of the two lineages with T. australiensis (GenBank) was well supported by bootstrap values (>70%), except for MP. The Australian Triops formed a cluster with T. longicaudatus (Fig. 3). Separate analyses of each mitochondrial genetic marker also produced the same tree topology to that of the combined datasets.

Discussion Individuals with ovisacs of both lineages exhibit differences in their external morphology. In lineage A, the anterior margin of the nuchal organ lies just in the posterior border of the interocular zone (Fig. 1D), the sulcus has 46–54 spines (Fig. 1F), and the dorsal-posterior border of the telson is spinose; in lineage B, the anterior margin of the nuchal organ crosses the posterior border of the interocular zone (Fig. 1G), the sulcus has 25–30 spines (Fig. 1I), and the dorsal-posterior border of the telson is smooth. Spencer and Hall (1895, 1896) described T. australiensis as having ~36 spines in the sulcus, contrasting with the specimens of lineages A and B. Unfortunately, the position of the nuchal organ and the posterior border of the telson were not mentioned by Spencer and Hall, and a comparison with the original specimens of T. australiensis is not possible, given that there is no type material of the species. Longhurst (1955a) placed four nominal species and all the Australian material that he examined into a single highly variable species, Triops australiensis, with high morphological variations: 35–43 total body rings, 5–12 legless body rings, and 28–62 spines in the sulcus of individuals with ovisacs. The mode of reproduction in natural populations is relevant to the definition and detection of different tadpole shrimp entities (e.g. species) (Sassaman 1991; Sassaman et al. 1997; Maeda- Martínez et al. 2000). The presence of testicular lobes within the ovary (ovotestes) has been taken as an indication of hermaphroditic reproduction (Bernard 1895; Longhurst 1955a, 1955b; Akita 1971). Ovotestes have been found in T. cancriformis from Europe, T. longicaudatus from California and Japan, and in several populations of Lepidurus (Longhurst 1955a, 1955b; Akita 1971; Zaffagnini and Trentini 1980). Spencer and Hall (1896) reported no evidence of development of Fig. 2. (A, B): Hermaphroditeof Triopscf. australiensis lineageB.(C): Male sperm in the ovaries of T. australiensis and Longhurst (1955a) of Triops cf. australiensis lineage A. (A): Longitudinal section (haematoxylin and eosin-stained) showing gonad tubules with no apparent testicular lobes. determined its reproduction as gonochoric (bisexual). We found (B): Transversal section (Fulgen picromethyl blue-stained) showing large no evidence of ovotestes in the three individuals with ovisacs testicular lobes in the haemocoel. (C): Transversal section (haematoxylin and representing Triops cf. australiensis lineage A. This finding and eosin stained) showing testicular tissue in the hemocoel. dl = digestive lumen; the presence of an almost equal number of males to individuals df = digestive tube; f = follicle; hc = hemocoel; st = somatic tubule; t = testis; with ovisacs in the specimens obtained suggest a gonochoristic tl = testicular lobes; vo = vitellogenic oocyte. reproduction mode. However, a more detailed study on testes functionality of Triops males remains to be done, given the existence of sterile males in other notostracans (Longhurst 1955b; 82 Australian Journal of Zoology G. Murugan et al.

Table 1. Genetic distances among Triops entities based on 12S and COI sequences estimated using the maximum composite likelihood model in MEGA 4.0 12S data are shown above the diagonal and COI data are shown below the diagonal

Taxon Triops cf. australiensis T. australiensis T. longicaudatus T. granarius T. cancriformis Lineage A Lineage B Mexico USA Japan Austria Triops cf. autraliensis Lineage A 0.034 0.021 0.073 0.070 0.098 0.142 0.125 Lineage B 0.085 0.026 0.062 0.059 0.104 0.129 0.120 T. australiensis 0.114 0.106 0.068 0.065 0.105 0.140 0.124 T. longicaudatus Mexico 0.127 0.147 0.128 0.008 0.109 0.136 0.126 USA 0.131 0.165 0.146 0.024 0.106 0.127 0.117 T. granarius 0.187 0.197 0.190 0.207 0.209 0.150 0.137 T. cancriformis Japan 0.199 0.176 0.167 0.187 0.191 0.251 0.013 Austria 0.219 0.185 0.183 0.196 0.200 0.261 0.021

Triops cf. australiensis lineage B mauritanicus) and T. granarius from four localities of Morocco. 72,83,80,99 Sassaman et al. (1997) also reported that an androdioecious 62,95,71,99 T. cf. australiensis lineage A Triops sp. (cited as Triops newberryi (Packard, 1871)) and a unisexual Triops sp.(cited as Triopslongicaudatus) were notonly 67,90,52,76 T. australiensis syntopic, but also had synchronic life cycles in pools in Arizona, USA, and that sexual populations of both taxa co-occurred T. longicaudatus (USA) at a site in Kansas, USA. According to Maeda-Martínez et al. 70,59,67,83 100,100, T. longicaudatus (Mexico) (1997), the probable factors contributing to the co-occurrence 100,100 of several large branchiopod species are related to habitat, T. cancriformis (Japan) species, or historic factors. Thiéry (1991) proposed four factors 100,100,100,81 100,100,100,100 as contributing to species assemblages: climatic and water T. cancriformis (Austria) characteristics favouring co-occurrence of naturally allopatric 100,91,96,100 species, diversity of food types may allow resource partitioning, T. granarius size differences, and horizontal or vertical segregation of species.

Lepidurus lemmoni Invertebrate assemblages and co-occurrence of anostracan species in the middle Paroo catchment is largely explained in Leptestheria dahalacensis terms of the influence of habitat factors, such as turbidity, salinity, and length of the wet phase (Timms and Boulton 2001; Timms Cyclestheria hislopi and Sanders 2002). 0.1 The genetic variation among populations of a species often depends on geographical distance: the smaller the geographic Fig. 3. Bayesian inference phylogram of Australian Triops based on COI distance, the more similar the DNA sequences (Penton et al. and 12S sequences. Numbers at nodes represent bootstrap values of maximum fi parsimony, neighbour joining,maximum likelihood, and posterior probability 2004). Conspeci c individuals of large branchiopods collected values of Bayesian inference. in one locality have been reported to show no or low genetic variation in the 12S and 16S rRNA genes (King and Hanner 1998; Remigio et al. 2003). However, populations collected at different Scanabissi and Mondini 2002). The four individuals with ovisacs places have been found to differ genetically. In T. cancriformis, of Triops cf. australiensis lineage B had large testicular lobes minor variation (0.2%, 12S) was observed among co-inhabiting within their gonads, indicating that they were anatomically individuals collected at three sites (Mantovani et al. 2004). hermaphrodites. This represents the first record of Australian Zierold et al. (2007) reported subspecies of T. cancriformis to Triops hermaphrodites, and suggests the occurrence of an vary by a maximum genetic distance (COI) of 3.5%. The androdioecious mode of reproduction (sensu Sassaman 1991, genetic differences found in this work between the two 1995; Sassaman and Weeks 1993) in tadpole shrimps of this Triops haplotypes and among these haplotypes and the continent. T. australiensis of the Paroo (Braband et al. 2002 [12S]; Richter Many cases of the association of two or more congeneric et al. 2007 [COI]) are not negligible. Even with respect to the species of large branchiopods in the same pool are known COI sequence of T. australiensis from an unspecified locality (see Maeda-Martínez et al. 1997; Timms and Sanders 2002). The (deWaard et al. 2006), haplotypes A and B showed a difference co-occurrence of lineages A and B does not represent a rare of 12 and 11%. phenomenon within the Notostraca. Thiéry (1991) recorded The two Triops entities from the Marsilea pool are distinct Triops mauritanicus (Ghigi, 1921) (cited as Triops cancriformis genetic lineages occurring in sympatry; they have different gonad Co-occurrence of two Triops cf. australiensis lineages Australian Journal of Zoology 83 anatomy (in individuals with ovisacs) and are distinguishable Korn, M., and Hundsdoerfer, A. K. (2006). Evidence for cryptic species in the by their external morphological characteristics. This suggests tadpole shrimp Triops granarius (Lucas, 1864) (Crustacea: Notostraca). reproductive isolation from each other. Thus, we conclude that Zootaxa 1257,57–68. Triops cf. australiensis lineage A and T. cf. australiensis lineage Korn, M., Marrone, F., Pérez-Bote, J. L., Machado, M., Cristo, M., Cancela da B probably represent two species that are distinct from Fonseca, L., and Hundsdoerfer, A. K. (2006). Sister species within the Triops cancriformis lineage (Crustacea, Notostraca). Zoologica Scripta T. australiensis. Further studies on tadpole shrimp systematics 35, 301–322. doi: 10.1111/j.1463-6409.2006.00230.x are still needed for a better understanding of the diversity and Linder, F. (1952). Contributions to the morphology and of taxonomy of Australian species of Triops. the Branchiopoda Notostraca, with special reference to the North American species. 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