Org Divers Evol DOI 10.1007/s13127-017-0343-5 ORIGINAL ARTICLE Revision of the genus complex Gibbula: an integrative approach to delineating the Eastern Mediterranean genera Gibbula Risso, 1826, Steromphala Gray, 1847, and Phorcus Risso, 1826 using DNA-barcoding and geometric morphometrics (Vetigastropoda, Trochoidea) Susanne Affenzeller 1,2 & Nicole Haar1 & Gerhard Steiner1 Received: 19 December 2016 /Accepted: 5 September 2017 # The Author(s) 2017. This article is an open access publication Abstract The trochoid genus, Gibbula, is abundant and di- morphometrics not only effectively delineated the sister gen- verse in the Mediterranean Sea but problematic to identify and era Steromphala and Phorcus but also delineated all analysed delineate. This is due to highly variable shell morphology, species in the Gibbula-Steromphala-Phorcus genus complex. vague original descriptions, and missing or unspecific type The additional use of geometric morphometrics enables re- material. In recent studies, COI barcoding yielded satisfactory searchers to compare barcoded material with fossil specimens results for species delineation. In the present study, a combi- or dry collections in an objective way. nation of geometric shell morphometric methods and COI barcoding was used to assess the most abundant species of Keywords Gibbula . Phorcus . Steromphala . Barcoding . the Eastern Mediterranean. All relevant identification charac- Geometric morphometrics . Species delineation ters were captured via standardised images of the shells in both lateral and ventral views. Agreeing with previous studies, Gibbula was recovered as paraphyletic in the molecular anal- ysis and thus is restricted to the clade encompassing the type Introduction species Gibbula magus (Linnaeus, 1758). The geometric mor- phometric analyses and the barcoding approach clearly distin- Gastropod taxonomy is traditionally shell-based. Methodology guish the remaining species into two groups: the genus in recent years using genetic data has revealed cryptic spe- Steromphala Gray, 1847 and the genus Phorcus Risso, cies and questionable species delineation in several gastro- 1826. Type material was used for the geometric morphometric pod taxa (e.g. Delicado and Ramos 2012; Weigand et al. analyses whenever possible. Based on re-examination of the 2013). Therefore, species identification and delineation by original type descriptions, lectotypes were designated. The shell morphology alone is often mistrusted if not supported joint application of DNA-barcoding and geometric by additional lines of evidence, such as DNA-barcoding or morphometric data. However, external characteristics (e.g. shell characters) remain the most straight forward way of identification of snails by scientists from related fields such Electronic supplementary material The online version of this article as ecology or palaeontology and especially by amateurs. (https://doi.org/10.1007/s13127-017-0343-5) contains supplementary Reliable species identifications and delineations of even material, which is available to authorized users. cryptic lineages not only are significant for taxonomic pur- * Gerhard Steiner poses but also impact the assessment of biodiversity, eco- [email protected] logical niche differentiation, and conservation measure- ments (e.g. Bálint et al. 2011; Feckler et al. 2014). Thus, 1 Department of Integrative Zoology, University of Vienna, it is of paramount importance—particularly for common Althanstraße 14, 1090 Vienna, Austria taxa—to re-evaluate the usefulness of shell morphology 2 Department of Geobiology, Georg-August-University Göttingen, for species delineation and identification, based on inde- Goldschmidtstraße 3, 37077 Göttingen, Germany pendent molecular markers. Mediterranean trochid S. Affenzeller et al. gastropods provide a suitable platform for re-evaluating et al. 2016) introducing several subgenera (e.g. Steromphala earlier hazy species descriptions and delineations in a con- Gray, 1847, Phorcus Risso, 1826, Gibbulastra Monterosato, temporary context. 1884) which later were discarded or revised again. Risso’s The systematics and phylogenetic relationships of the tro- (1826) original species allocations to the two genera, Gibbula choid families Trochidae and Turbinidae and the subfamily and Phorcus, were later re-established (Thiele 1929-35; Cantharidinae are only partly resolved (Williams and Ozawa Nordsieck 1968; Gofas and Jabaud 1997). Phylogenetic and 2006; Williams et al. 2008, 2010). Although molecular data systematic relationships remain unresolved. support the monophyly of the Cantharidinae (Williams et al. 2010; Uribe et al. 2016), relationships within the subfamily Shell morphometrics remain ambiguous. One of the subfamily’s most problematic groups contains Morphometric methods have been successful in identifying Gibbula Risso, 1826 and Phorcus Risso, 1826. Problems with certain species of gastropods via analysis of shell form correctly identifying Gibbula and Phorcus species are main (Kirchner et al. 2016). Since the classical morphometric mea- reasons for this unresolved status. An umbilicated shell, black sures like lengths and angles introduced by Thompson (1917) and white striped epipodial tentacles, a non-calcified opercu- are insufficient to capture shape differences in the present taxa, lum, and a turbinate shell form currently characterise both geometric morphometric approaches utilising landmark-based genera. Species differ in spiral height, umbilicus size and algorithms are a valuable alternative. Mathematically, they are shape, and overall shell form. Shell sculpture is diagnostic increasingly applied to assess form and shape as well as vari- for a few species, and even coloration is sometimes used in ation and intended as an objective means of comparison (Rohlf the literature. However, all these characters show such a wide 1998). Landmarks and semi-landmarks offer the possibility to range of variation that reliable identification of species to analyse objects without losing their geometrical form in the Gibbula and Phorcus is difficult and depends on subjective, process (Bookstein 1991; Mitteroecker and Gunz 2009). personal experience (e.g. Barco et al. 2013). Gastropod shells offer only few useful morphological land- Among the reasons for the confusing status of some of marks as defined by Bookstein (1991). Thus, it is necessary these species are their vague original descriptions, mostly to use semi-landmarks on outlines, which are processed later based on dry and empty shells. To exacerbate the situation, on in the sliding landmark algorithm, allowing for the land- some of the type collections have been destroyed or lost mark to slide on a tangent connection between neighbouring (Table 1), making re-descriptions impossible. Other type col- landmarks in order to optimise the Procrustes fit (Bookstein lections contain many syntypes, some of which do not show 1997; Mitteroecker and Gunz 2009). Cartesian coordinates consistent morphology. The vagueness of original descrip- gained from landmarks and semi-landmarks are aligned in a tions leads to problems in recent literature used for identifica- generalised Procrustes fit. Through a stepwise process, the tion, e.g. for the identification of G. varia, G. rarilineata and original landmark configuration of each individual is scaled juvenile G. divaricata (Gofas et al. 2011; Nordsieck 1968), as and rotated in a way to gain a group of landmark configura- well as G. umbilicaris and G. nebulosa (Barco et al. 2013; tions showing the least possible difference. In this process, Oliverio pers. comm.). each resulting landmark configuration therefore, independent In the comprehensive phylogenetic study by Donald et al. of size, orientation, and position, only comprises shape in the (2012), the monophyly of Gibbula, represented by G. magus, end (Bookstein 1996). The resulting Procrustes coordinates G. fanulum, G. cineraria, G. pennanti, G. rarilineata, define the shape of an object in the Kendall shape space G. umbilicalis,andG. varia, is disrupted by species of (Kendall 1981, 1984). Although its surface is curved, Osilinus, a junior synonym of Phorcus (Donald et al. 2012), Procrustes distances can be approximated by Euclidean dis- and Williams et al. (2010)byJujubinus ( See Table 1 for taxon tances in the tangent space and can thus be analysed statisti- authorities). In the most recent molecular study based on six cally through multivariate methods like principal components different genes (both mitochondrial and nuclear) by Uribe analyses (Mitteroecker and Gunz 2009). Geometric morpho- et al. (2016), the monophyly of Gibbula is disrupted by both metrics, thus, offers the possibility of analysing and comparing Phorcus and Jujubinus. In the most comprehensive molecular complex shapes of shells of different sizes in a single sample study on the genus Gibbula, Barco et al. (2013) resolved the (Mitteroecker and Gunz 2009). dilemma by using Phorcus species as outgroup and omitted other cantharidinid genera such as Jujubinus, whereby Aims of the study Gibbula was recovered monophyletic. The morphological and phylogenetic problems in Gibbula The genera Gibbula and Phorcus have undergone consider- and Phorcus (Philippi 1836-44, 1849;Thiele1929-35; able changes in their 190 years of history. The rather vague Nordsieck 1968) and their taxonomic implications were ad- morphological diagnosis of Gibbula and Phorcus left ample dressed by different authors (e.g. Williams et al. 2010,Uribe room for species assignment to these