Significance of morphological and mitochondrial characters in species delimitation
The most consistent and reliable character for species delimitation in every group was the internal anatomy of the penis, along with other genital characters relating to the penis, epiphallus and spermatophore. The sculpture of the penis interior was informative at both genus and species level, with a general pattern observed within a genus (for example, longitudinal pilasters and the presence of a penial verge in Mysticarion) and a unique, highly consistent anatomy in each species (for example, number and shape of longitudinal pilasters, size and shape of penial verge). Similarly, the spermatophore provided useful characters at both genus and species level; however, the small number of spermatophores collected limited the value of this character somewhat. Variation in the spermatophore is reflected in the shape of the flagellum, and although less detail is available, in some cases differences in the flagellum shape were sufficient to infer differences in the spermatophore.
Penis morphology shows great variation among land snails and is generally species-specific, supporting the hypothesis that it is an important factor in species recognition during copulation (Gómez, 2001). It is likely that the spermatophore plays a similar role (Gómez, 2001). In the related slug family Milacidae, spermatophores are also heavily sculptured with branching spines and hooks, and are highly species-specific, in some cases providing the most useful means of species discrimination (Wiktor, 1987).
Body colour and shell dimensions, which are of high value in rapid, non-expert identification, were also informative characters in species delimitation in many groups. Body colour was consistent within species, although preserved specimens were faded, making colour markings sometimes difficult to accurately assess. In particular, Mysticarion obscurior, which co-occurs with congeners M. porrectus and M. insuetus and was often misidentified in museum collections, could reliably be distinguished in life by its darker body colour and in faded alcohol-preserved specimens by the disjunct black markings on the right shell lappet. Even in alcohol-preserved specimens body colour could be used to distinguish species in Cucullarion and Brevisentis, but was less informative in Parmavitrina, where colour differences are more subtle.
Characters relating to the shell, commonly used in land snail identification, are now thought to be significantly influenced by environmental factors and as a result can show high levels of both convergence and conservatism (Stankowski, 2011; Criscione and Köhler, 2013a; Köhler and Criscione, 2015). Within the south-eastern Australian helicarionid clade there is great diversity in shell form, ranging from snails with a complete shell to semislugs with a highly reduced shell. Gross shell shape was therefore an important character in the separation of genera, although convergence was evident in (for example) the semislugs Peloparion helenae and Ubiquitarion iridis. At the species level, differences in shell size and shape (primarily height-to-diameter ratio) were informative, particularly in Brevisentis, Mysticarion and Parmavitrina, although differences were slight and in many cases could not reliably be used as a sole means of identification. Protoconch microsculpture has been used to delimit genera in many Australian land snails including the Helicarionidae (Stanisic et al., 2010); however, we did not find significant differences in shell microsculpture, with the exception of a unique sculpture of notched spiral grooves on the protoconch of Mysticarion.
Species delimited by their morphology were consistently found to form monophyletic clusters in the mitochondrial trees, which usually were well separated from each other by long basal branches. The amounts of interspecific genetic differentiation observed here were at the lower end of the rather wide spectrum of average interspecific differences among stylommatophoran land snails (Davison et al., 2009; Criscione et al., 2012; Köhler and Johnson, 2012; Criscione and Köhler, 2013b; Burghardt and Köhler, 2014). In addition, there has been considerable overlap between the ranges of intraspecific and interspecific distances in the southeastern Australian helicarionids, particularly in 16S (Fig. 5). This overlap is attributable primarily to the high intraspecific variation in the widespread Ubiquitarion iridis, and the comparatively low interspecific variation among P. megastoma, P. flavocarinata and P. maculosa. However, we consider the differences in the penial complex as sufficient to justify their separation as distinct species, particularly given that two of them occur in sympatry with each other. The particularly low interspecific distances among these three species are probably indicative of their rather recent origin.
The highest intraspecific differences among the studied species were observed in Ubiquitarion iridis (COI 2.8%, 16S 2.2%) and Brevisentis atratus (COI 2.8%, 16S 1.6%). In neither case was this reflected by any morphological differentiation nor were these distances remarkable when compared with other land snail groups (e.g., Davison et al., 2009). In contrast to the abovementioned species, we found that the two sympatric species of Cucullarion exhibit a high degree of genetic distinctiveness while being distinguished by rather subtle anatomical differences.
Our findings of such inconsistent amounts of morphological and mitochondrial differentiation among the examined species confirm that species are best delimited by combining the appraisals of their morphological and mitochondrial differentiation.