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Sister species within Triops cancriformis • M. Korn et al.

Table 2 Overview of the sequences retrieved from the GenBank for the DNA was precipitated for 30 min at −20 °C and pelleted by
this study with their accession numbers. Lepidurus cryptus (Rogers centrifugation at 4 °C. The DNA pellet was washed twice with
2001) refers to the cryptic lineage found by King & Hanner (1998). ice-cold 70% ethanol, dried and dissolved in 50 µL TE buffer.

Part of the 16S rDNA gene was amplified using the universal
Taxon Accession numbers Gene
primers 16Sar and 16Sbr (Palumbi et al. 1991; corresponding
T. cancriformis AB084514 16S + 12S to primers 16SA and 16SB from Simon et al. 1994) and
T. cancriformis AY115613, AY159571–579 16S the 12S fragment with the universal primers published in
T. numidicus AF200963–971, AY115612 16S Colbourne & Hebert (1996) and Murugan et al. (2002). Each
T. longicaudatus AY639934, AY115605, AY115609, 16S
PCR was performed with about 500 ng template DNA in a
AY115610–611, AY159580–581
50-µL volume (5 pmol of each primer, 1.25 nmol of each
L. a. apus AY159584 16S
L. a. lubbocki AY159582–583 16S dNTP, and 0.2 u of Taq polymerase (Bioron) buffered with
L. arcticus AY159585 16S 10 mM Tris-HCl, 50 mM KCl, 0.5% Triton X-100, 1.5 mM
L. lemmoni AY115614 16S MgCl ). Following the initial 4-min denaturation at 94 °C,
T. cancriformis AY115603, AY159563–65, AF494482 12S 2
the program consisted of 35–50 cycles (depending on DNA
T. numidicus AY115602 12S
quantity) of 45 s at 94 °C, 60 s at 55 °C, 90 s at 72 °C, and
T. australiensis AY050646 12S
T. longicaudatus AY115595–600, AY159566, TLAJ0817, 12S 5 min at 72 °C for final elongation. For sequencing, the PCR
AY639934, AY115601 products were sent to the DNA Sequencing Facility of the
L. a. apus AF494483, AY159568 12S Max Planck Institute of Molecular Cell Biology and Genetics
L. a. lubbocki AY159567 12S
(Dresden, Germany). The forward primers were used for
L. arcticus AY159569 12S
direct sequencing of the PCR product, and if the sequence
L. lemmoni AY115604, LLAJ0823–824, LLAJ0830 12S
L. packardi LPAJ0820–822 12S was not of sufficient quality the complement/reverse sequence
L. couesii LCAJ0827 12S was obtained additionally.
L. cryptus LCAJ0819, LCAJ0825–826, LCAJ0829 12S
L. bilobatus LBAJ0818, LBAJ0828, LBAJ0831 12S
Sequence alignment, nucleotide composition, and

substitution patterns
Alignment was performed by hand using the program
DNA extraction, PCR amplification and sequencing BioEdit (Hall 1999). In the 16S dataset one Lepidurus and one
For total DNA extraction, a piece of the thoracopod (or the Triops longicaudatus sequence were included as outgroups. In
whole specimen if it was very small) was washed twice with the ingroup we encountered only one insertion in the 16S
100 µL TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) gene (an additional A at the position 272 in the unequivocal
and then placed into 500 µL DTAB buffer (65 °C; 37.5 mM alignment); additional gaps had to be introduced at three
EDTA, 1.125 mM NaCl, 75 mM Tris-HCl, pH 8.0) in an positions with respect to the outgroups. Two deletions and
Eppendorf cup. The material was crushed with a small pestle two insertions had to be introduced within the ingroup in the
or cut into small pieces with scissors and incubated for 30 min 12S dataset (alignment available upon request). Nucleotide
at 65 °C. After addition of Proteinase K (0.5 mg/sample), the composition, substitution frequencies, pairwise transition/
extractions were incubated overnight at 55 °C. To remove transversion frequencies, and pairwise distances were
surplus RNA, the samples were incubated with 0.1 mg calculated with PAUP* 4.0b10 (Swofford 1998). To enable
RNAase A for 30 min at 37 °C, after which the extracts were an assessment of the overall range of intrageneric sequence
cleaned with chloroform/isoamyl-alcohol (24 : 1) twice. After divergence found among recognized species of Notostraca,
addition of 1/5 volume 4 M LiCl and 1 volume isopropanol, we compared the mean genetic distances between congeneric

Table 3 Literature data (Longhurst 1955) used for the classification of subspecies of Triops cancriformis. Longhurst (1955) notes that it may be
difficult to ascribe single specimens (with a smooth carina) to the nominate race or T. c. simplex, but he found no large samples of T. c. cancriformis
without specimens showing carina spines. To date, it still is not clear if the maleless ‘hermaphrodite’ populations reproduce by selfing or by
parthenogenesis. There are also populations known that are strongly female/hermaphrodite dominated.

Character T. c. cancriformis T. c. simplex T. c. mauritanicus
Carina spines 0–10, generally 2–3 complete absence, more numerous and much
carina ‘quite’ smooth stronger than in T. c. cancriformis
Size of furcal spines small small very large
No. of apodous segments of female 4–6 5–7 5–7
Reproductive mode hermaphrodite and bisexual bisexual bisexual

304 Zoologica Scripta, 35, 4, July 2006, pp301–322 • © 2006 The Authors. Journal compilation © 2006 The Norwegian Academy of Science and Letters