Zoologische Mededelingen, 83 (July 2009)
Molecular phylogenetic relationships of Inchoatia taxa
D.R. Uit de Weerd 1, D. Schneider 2, E. Gittenberger 3
Keywords: Gastropoda, Clausiliidae, Inchoatia, Greece, molecular phylogenetics.
The molecular phylogenetic relationships between nominal (sub)species within the Greek land snail genus Inchoatia are examined.
The genus Inchoatia was introduced by Gittenberger & Uit de Weerd (2006a, 2006b) on the basis of molecular phylogenetic analyses (Uit de Weerd et al., 2004). In its present form the genus consists of four nominal species: I. haussknechti (O. Boettger, 1886), I. inchoata (O. Boettger, 1889), I. megdova (H. Nordsieck, 1974) and I. regina (H. Nordsieck, 1972). Besides synapomorphic nucleotide substitutions, these four taxa share a similar shell shape and have more or less continuous ranges. According to the molecular data, the major division within Inchoatia is between (1) an I. inchoata + I. regina subclade, and (2) an I. haussknechti + I. megdova subclade. This pattern is congruent (1) with genital anatomical differences between the two subclades, previously used to place them in separate genera, viz. Sericata and Carinigera (see Nordsieck, 1974), and (2) with conchological similarities between the species within each subclade.
A major problem has been the morphological and geographical delimitation of the species within each subclade. Nordsieck (1972) recognizes I. regina as a separate species with a range situated amidst those of the I. inchoata subspecies, stating (p.16) that the differences with I. inchoata are sufficiently large (“groß genug”). Similarly, I. haussknechti and I. megdova have a mosaic distribution. However, where their populations come geographically close, without contact zones as far as known, morphological convergence is not seen. For more details, see Gittenberger & Uit de Weerd (2009).
The molecular phylogenetic study by Uit de Weerd et al. (2004) was not designed to answer questions about the status of the speciesgroup taxa within Inchoatia. Only four samples, representing each of the four nominal species, were included: I. haussknechti alticola (H. Nordsieck, 1974), I. inchoata inchoata (O. Boettger, 1889), I. megdova tavropodensis (Fauer, 1993), and I. regina (H. Nordsieck, 1972). Moreover, according to Nordsieck (2007: 109), “[I.] haussknechti alticola H. Nordsieck and [I.] megdova tavropodensis Fauer, belong to one and the same species”. Referring to an unpublished revision, Nordsieck (2007) recognizes not two, but four species within the I. haussknechti - I. megdova assemblage (see Table 1). Thus the diversity within this assemblage may have been insufficiently sampled by Uit de Weerd et al. (2004).
As a step towards a more objectively founded taxonomic and biogeographical delimitation of taxa within Inchoatia, we determined cytochrome c oxidase subunit 1 (COI) nucleotide sequences of multiple samples of seven additional subspecies within the Inchoatia clade, and conducted phylogenetic analyses.
Materials and methods
In order to unravel the interrelationships among the taxa within the genus, we supplemented the four Inchoatia sequences of Uit de Weerd et al. (2004) with 19 additional partial cytochrome c oxidase subunit 1 (COI) sequences (see Table 2), following the same procedures. With this increased sampling all three subspecies of I. inchoata are represented, as are three subspecies of I. haussknechti and three subspecies of I. megdova (see Gittenberger & Uit de Weerd, 2009). As an outgroup four previously determined partial COI sequences (AY425562-AY425565) of the closely related (Uit de Weerd et al., 2004) genus Cristataria were used. All phylogenetic analyses were performed using PAUP* 4.0b10 (Swofford, 2002). Prior to the analyses, we made certain that the base frequencies for each of the three codon positions were non-inhomogeneous across taxa (Chi-square test). Heuristic MP searches consisted of 1000 random addition replicates (tree bisection and reconnection, steepest descent). In addition, an MP bootstrap analysis consisting of 10,000 replicates was performed. Each replicate consisted of 10 random addition replicates (tree bisection and reconnection, steepest descent).
Table 2. Information on the populations sampled as an ingroup in the phylogenetic analyses of the genus Inchoatia. (*) COI sequences obtained by Uit de Weerd et al., 2004. For information regarding the outgroup samples, see Uit de Weerd et al., 2004.
Results and discussion
The phylogenetic analyses retrieved two MP trees (score 562), the strict consensus of which is shown in figure 1. This tree confirms that Inchoatia consists of two main subclades, i.e. subclade (1) consisting of Inchoatia inchoata en Inchoatia regina, and sub-clade (2) with Inchoatia haussknechti and Inchoatia megdova.
Fig. 1. Strict consensus of two MP trees for the COI dataset. Numbers along branches indicate bootstrap values. Encircles numbers refer to subclade 1 and subclade 2. GenBank accession numbers are given behind taxon names.
Within subclade 1, Inchoatia regina is nested in I. inchoata. As a sistergroup to Inchoatia inchoata klemmi (H. Nordsieck, 1972), I. regina constitutes two successive highly (≥ 99% bootstap) supported clades with I. inchoata subspecies. This result is congruent with the conchological similarities between I. regina and I. inchoata klemmi, which are also biogeographical neighbours. We therefore see no reason to attach a special status to I. regina as a separate species next to I. inchoata, and propose to classify this taxon as Inchoatia inchoata regina.
In subclade 2, the phylogenetic status of the species Inchoatia megdova and I. haussknechti is far less clear. Although the COI sequences of the I. megdova megdova population are nested among the I. haussknechti sequences, this nested position is comparatively weakly supported (bootstrap value 74% and 65%). The remaining two I. megdova subspecies sampled are placed on separate branches within a basal trichotomy in subclade 2.
The three most parsimonious trees (score 576) congruent with the classification suggested by Nordsieck (2007) are significantly (one-sided Templeton test: P<0.01) less supported by the COI data than either MP tree. This is not consistently the case (one-sided Templeton test: P = 0.11; P = 0.04) for the most parsimonious tree (score 568) congruent with the prevalent classification, in which I. megdova and I. haussknechti are both monophyletic and together accommodate all subspecies within the assemblage, including I. megdova bruggeni Gittenberger & Uit de Weerd, 2009. In the absence of evidence to the contrary, we therefore propose to maintain that latter classification.
Edited: A.S.H. Breure
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