During the course of a project aiming at the reconstruction of the colonization of the South Pacific islands by tateid gastropods based on molecular data we discovered five new species on New Caledonia belonging to the genera Hemistomia and Leiorhagium, respectively. We describe these species based on morphological, anatomical and genetic data. All five species are morphologically cryptic as they closely resemble or are even indistinguishable from known species stressing the importance of a comprehensive taxonomic approach integrating several methods. As a consequence of their small and fragmented geographic ranges and the rapidly progressing anthropogenic land cover changes on New Caledonia, all five species qualify as critically endangered according to the criteria of the IUCN.

Conservation, cryptic species, endemic, integrative taxonomy, IUCN, New Caledonia, South Pacific, spring snails, Tateidae

New Caledonia is famous for being a biodiversity hotspot harboring a high number of endemic species (Myers et al. 2000) including a radiation of small freshwater gastropods belonging to the family Tateidae. This radiation is probably of Oligocene origin and comprises more than 50 species in seven genera (Haase and Bouchet 1998, Zielske and Haase 2015). Many of these species are extreme narrow-range endemics known from only few or single sites (Haase and Bouchet 1998), a pattern typical for Truncatelloidea in freshwaters worldwide (e.g. Giusti and Pezzoli 1980, Radoman 1983, Haase 1996, 2008, Ponder and Colgan 2002, Liu and Hershler 2005, Hershler et al. 2011, Delicado and Ramos 2012). In the frame of a project aiming at the reconstruction of the colonization of the South Pacific islands by tateids based on molecular data (Zielske and Haase 2014a, b, 2015, Zielske, Ponder and Haase in preparation) we visited New Caledonia in May 2012 in order to collect suitable material for sequencing. During this expedition we found five new species of the genera Hemistomia Crosse, 1872 and Leiorhagium Haase & Bouchet, 1998, respectively (Figs 1, 2), which we describe herein based on morphological, anatomical and genetic data. All five species qualify as morphologically cryptic as they closely resemble or are even indistinguishable from known species (see Pfenninger and Schwenk 2007). The discovery of new cryptic species was predicted by Haase and Bouchet (1998), whose revision was based solely on morphology and anatomy. In general, cryptic species are common among different spring snail families of Truncatelloidea (e.g., Liu et al. 2003; Haase et al. 2007; Delicado and Ramos 2012; Collado et al. 2013).

Figure 1. 

Localities of new species and samples used for morphometric comparisons. Inset shows position of New Caledonia in the Southwest Pacific. Arrows indicate type localities of species represented by more than one sample (see also Table 1).

Figure 2. 

Holotypes. A Hemistomia andreae sp. n. B Leiorhagium adioincola sp. n. C Leiorhagium aremuum sp. n. D Leiorhagium clandestinum sp. n. E Leiorhagium neteae sp. n. Scale bar = 1 mm.

Snails were fixed in 70% ethanol in the field, transferred to propylene glycol for shipping by courier, and returned to ethanol, this time 96%, after arrival in our lab. For measurements, up to 20 snails per sample were photographed under a Zeiss SteREO Discovery.V20 dissecting microscope with a Zeiss Axio Cam MR3. Five dimensions – shell height, shell width, aperture height, aperture width, body whorl width – were measured using the program AxioVision 40 V4.8. (Zeiss) and whorls counted to the nearest eighth (Kerney and Cameron 1979). Up to six shells were dissolved in diluted hydrochloric acid for dissections. Anatomies were photographed as well. These digital images served as template for drawings made on a graphical tablet. For scanning electron microscopy up to three shells, radulae and opercula were cleaned in 5% sodium hypochlorite. The cephalopodia of up to two males were dried using hexamethyldisilazane (Nation 1983). After sputter coating with gold objects were investigated in a Zeiss EVO LS10 Scanning Microscope.

Morphometric analyses of shell measurements including canonical variates analyses (CVA) maximizing the differentiation of groups in multivariate space, multivariate analyses of variance (MANOVA), assignment tests, and Hotelling’s T²-tests were conducted in PAST 2.12 (Hammer et al. 2001). Sequential Bonferroni-correction was applied to multiple tests. These analyses also included samples of known, similar species the new ones could be mistaken for (Table 1). The selection of species used in comparisons was based on the phylogenetic analysis.

Phylogenetic analyses were based on a selection of sequences generated by Zielske and Haase (2015), who analyzed fragments of the mitochondrial genes cytochrome oxidase subunit I (COI) and 16S rRNA as well as the nuclear internal transcribed spacer 2 (ITS2). For lab protocols see Zielske and Haase (2014a, 2015). We restricted the analysis to 3 specimens per species at most and used Kanakyella gentilsiana, Crosseana crosseana, and C. melanosoma as outgroups (Table 1). The alignment of 16S rRNA and ITS2 was generated using secondary structure information using RNAsalsa 0.8.1 (Stocsits et al. 2009) (for details see Zielske and Haase 2015) and checked for ambiguous and randomly similar sites in Aliscore 2.0 (Misof and Misof 2009). We defined seven partitions. PartitionFinder 1.1.0 (Lanfear et al. 2012) identified the following scheme and substitution models as optimal among all possible combinations of separate and merged partitions: COI 1^st positions (TrNef+I), COI 2^nd positions (F81), COI 3^rd positions (TVM+I+Γ), 16S rRNA loops (TrN+ Γ), ITS2 loops (TrNef+I+Γ), joint stems of 16S rRNA and ITS2 (K80+I). With these settings, tree reconstructions were conducted in a maximum likelihood (ML) framework using GARLI 2.01 (Zwickl 2006) with 500 replicates. Robustness was assessed by bootstrapping with 200 replicates.

Type and non-type material is deposited at the Museum National d’Histoire Naturelle in Paris (MNHN) and at the Naturhistorisches Museum Wien(NHMW).

Our phylogenetic analyses based on DNA sequence data confirmed the suspicion of Haase and Bouchet (1998) that additional cryptic species in this snail fauna will be identified once molecular methods are applied emphasizing the huge morphological variability of certain nominal species. Recent accounts on tateid gastropods from Vanuatu and Fiji (Zielske and Haase 2014a, b) have revealed extensive radiations of morphologically very similar species. However, in contrast to the New Caledonian taxa, the radiations on those archipelagos are comparatively young (Zielske and Haase 2015). Four of the five species described here are hardly distinguishable from known taxa based on measurements despite being genetically well differentiated with even uncorrected distances (see Fregin et al. 2012) of at least 7.4% to their next similar congeners. Whether this means that morphologically similar species occupy similar niches is impossible to tell at this stage because the relationship of shell morphology to habitat has not been investigated among truncatelloidean gastropods except for a few accounts on Potamopyrgus antipodarum (Haase 2003, Holomuzki and Biggs 2006, Kistner and Dybdahl 2013). Although ranges overlap or are contiguous, sibling species have not (yet) been encountered in sympatry, i.e. in the same spring or stream.

The new species provide an additional truncatelloid example stressing the importance of an integrative taxonomic approach combining morphological, anatomical and genetic methods (e.g. Haase et al. 2007, Delicado and Ramos 2012). Given the mosaic nature of evolution of these small gastropods with morphologically as well as genetically cryptic species (e.g., Haase et al. 2007, Haase 2008, Zielske et al. 2011, Delicado and Ramos 2012, Liu et al. 2013), we do not adhere to a fairly strict scheme of species identification as advocated elaborately e.g. by Schlick-Steiner et al. (2010). Instead we advocate the approach of Padial and de la Riva (2010) who have a more natural vision of the evolutionary processes potentially involved in speciation. For instance, they acknowledge that the congruence of different character sets, pivotal for taxonomic decisions for Schlick-Steiner et al. (2010), may be plesiomorphic.

Genetic differentiation was an important indicator of species status. Pairwise p-distances > 7.4% are far above any threshold suggested by advocates of barcoding (e.g., Hebert et al. 2003, 2004; Ratnasingham and Hebert 2007). However, again we do not adhere to a strict scheme as there may be no mitochondrial differentiation between good species as well as considerable variation within species of spring snails (e.g. Haase 2008; Zielske et al. 2014a; see also Fregin et al. 2012). That genetic differentiation does reflect species status for the new taxa is also indicated by the comparison of their branch lengths to branch lengths among morphologically well defined species in our phylogenetic analysis.

While conducting our morphometric analyses we appreciated that the measuring methods applied for the material described previously (Haase and Bouchet 1998) and for this account are incompatible. Obviously, using an ocular micrometer fitted to a dissecting microscope produced inaccurate data, although the measurements were quite consistent judging from the fairly low coefficients of variation, which were of a similar order of magnitude as those computed for the present data. Therefore, we had to re-measure the old samples used in our comparisons.

Another methodological problem almost expectedly occurred in the field. All collections made for our previous monograph (Haase and Bouchet 1998) were geo-referenced from maps. This proved to be fairly inaccurate when we tried to relocate sites in 2012 guided by GPS. Additional difficulties arose from recent road development and land-use changes. Many villages are now accessible on much broader roads than 20 years ago. Construction has obviously destroyed small road-side springs and seepages and changed the course of streams. Other sites were destroyed by extensive fires affecting entire valleys or hills. Crosseana melanosoma, in our analysis part of the outgroup, used to be common when first collected in 1992. Now we found only a few specimens. It remains to be seen whether there are other (extant) populations in the unexplored hinterland of Boyen. In contrast, H. yalayu, collected in a few seepages on Col d’Amoss in the far Northeast in 1989, is now probably extinct. The entire area has lost its primary vegetation. Today, the fire resistant niaouli (Melaleuca quinquenervia) and shrubland are dominating and streams harbor a very depauperate fauna.

Four of the five new species were found in single sites and the fifth was found at only two sites. Considering the vulnerability of small habitats like springs and the rapid anthropogenic development and changes on New Caledonia just outlined immediately raises concern regarding the chances of long-term survival of these species (see also Haase et al. 2010). Most sites we surveyed were rather easily accessible, close to roads, so that one can assume that there are other populations deeper in the forests or forest remnants. Nevertheless, given that the area of occupancy of each species is certainly less than 10 km², that ranges of spring snails are almost naturally severely fragmented, and the rapidly progressing change of land cover, areas of occupancy as well as habitat, hence the numbers of populations will decline. Therefore, all five species and probably the majority of New Caledonian tateids qualify as critically endangered according to the criteria (CE, B2,a,II-IV) of the International Union for Conservation of Nature (IUCN 2012).

This publication would not have been possible without the assistance of numerous local field guides and landowners who provided access to their properties. We thank Christel Meibauer for assistance in the molecular lab. Rabea Schlüter is acknowledged for support at the SEM. We are grateful to the authorities of New Caledonia for issuing the relevant permits. Christine Pöllabauer has been a generous host in Noumea during our field trip. The manuscript benefited from suggestions of two anonymous reviewers and Robert Hershler. Financial support was received from the German Research Foundation (HA 4752/2-1).