Morphological comparisons reveal detailed similarities between Procambarus fallax and Marmorkrebsnext section
Concerning morphological characters the Marmorkrebs identifies closely with P. fallax and is quite distinct from P. alleni (Figs 2, 3). The Marmorkrebs annulus ventralis lacks the characteristic scooped ‘wings’ on the lateral parts and the anterior portion is not peaked like the annulus ventralis of P. alleni (Fig. 2A vs. B-C). In contrast, the Marmorkrebs annulus ventralis closely resembles the flatter, bell-shaped annulus ventralis of P. fallax (see also Hobbs, 1942; Kawai et al., 2009) (Fig. 2). Furthermore, the Marmorkrebs annulus ventralis does not closely resemble that of any other North American members of the genus Procambarus (Hobbs, 1989). The overall coloration pattern of P. fallax (Fig. 3), including a ‘marbled’ carapace and lateral dark stripes, is consistent with the coloration of Marmorkrebs from the Berlin laboratory culture, in published photos (Scholtz et al., 2003; Seitz et al., 2005; Vogt et al., 2008; Kawai et al., 2009), and with those on the internet. The dark lateral horizontal stripes through the cephalothorax and abdomen on P. fallax (Fig. 3B) can vary in intensity between individuals (or populations), but a distinct stripe is a good indication that the crayfish is P. fallax (Fig. 3B) rather than P. alleni (Fig. 3A). Specimens of Procambarus alleni have conspicuous dark spots ventral to the bases of the eye stalks and anterior to the oral cavity (Fig. 3A) while those of P. fallax do not (Fig. 3B). This was first pointed out by Hendrix and Loftus (2000) and after seeing thousands of individuals of these species we can further attest to the uniformity of this difference in south Florida (N. Dorn, pers. obs.). All the Marmorkrebs individuals studied in the laboratory culture in Berlin lack these conspicuous spots (Fig. 3C). The absence of these spots can also be clearly seen in Kawai et al. (2009) from Marmorkrebs from the Japanese aquarium trade (Fig. 3D).
Fig. 3. Photographs of live A) Procambarus alleni and B) Procambarus fallax from south Florida, USA and C) Marmorkrebs from the lab cultures in Germany and the aquarium trade in Japan D). In every case the lateral overall view (left) and the facial ventral view (right) is shown. The arrows in the left pictures mark the dark lateral horizontal stripes through the cephalothorax and abdomen in P. fallax and Marmorkrebs (compare with Fig. 1). The arrow in the top right picture points at the facial dark spots characteristic for P. alleni.
Molecular analysis shows a high degree of DNA sequence correspondence between Procambarus fallax and Marmorkrebs
The molecular analysis shows distances of the COI sequences (total length 675 base pairs) between P. alleni and P. fallax ranging from 6.40 to 7.24% (mean 6.88 ± 1.01%), between P. alleni and Marmorkrebs from 6.75 to 7.24% (mean 6.88 ± 1.05%), and between P. fallax and Marmorkrebs from 0.60 to 0.75 (mean 0.67 ± 0.24%). Within the species, the values are 0.00-1.36% (mean 0.79 ± 0.25%) in P. alleni and 0.00-1.05% (mean 0.57 ± 0.19%) in P. fallax. The 12S sequences (381 base pairs) of P. fallax and Marmorkrebs are identical. This is also true for the P. alleni specimens, where uniform sequences are found except in specimen pal-10, which shows a single substitution. The distance between the P. fallax / Marmorkrebs complex and the P. alleni population ranges from 2.95 to 3.27% (mean 3.02 ± 0.92%). The two Marmorkrebs specimens are identical in the COI and 12S sequences over a total length of 1057 base pairs (see also Martin et al., 2010).
For tree reconstruction, a total data set consisting of the combined COI and 12S sequences of the specimens listed in Table 2 and a selection of several species from GenBank® of 927 nucleotides was available after pruning the DNA alignments with the program Gblocks 0.91b. The resulting tree shows that the two Marmorkrebs specimens nest clearly within the P. fallax representatives (Fig. 4). The same result was obtained if each gene alone was used for tree reconstruction (not shown).
Fig. 4. Maximum-likelihood analysis of Marmorkrebs, several Procambarus species, and Orconectes limosus as outgroup. The tree was estimated from combined nucleotide sequence data sets of COI and 12S mitochondrial genes calculated under the J1+G and the HYK+G model, respectively, with five rates categories. Numbers above branches are values in percent obtained from the bootstrap analysis of 1,000 replicates (bootstrap support values lower than 50% are not shown). The scale bar indicates the evolutionary distances in substitutions per site. Accession numbers of the used sequences from the GenBank© are (COI /12S) (÷ = sequence not available): P. clarkii AY701195/EF 012280, P. acutus AF474366/FJ619794, P. curdi Reimer, 1975 ÷/EF012281, P. liberorum Fitzpatrick, 1978 ÷/EF012311, P. nigrocinctus Hobbs, Jr., 1990 ÷/EF012282, P. reimeri Hobbs, 1979 ÷/EF012283, P. simulans (Faxon, 1884) EU583575/÷, O. limosus AY701199/AY151531.