Uncovering the shell game with barcodes: diversity of meiofaunal Caecidae snails (Truncatelloidea, Caenogastropoda) from Central America

Abstract Caecidae is a species-rich family of microsnails with a worldwide distribution. Typical for many groups of gastropods, caecid taxonomy is largely based on overt shell characters. However, identification of species using shell characteristics is problematic due to their rather uniform, tubular shells, the presence of different growth stages, and a high degree of intraspecific variability. In the present study, a first integrative approach to caecid taxonomy is provided using light-microscopic investigation with microsculptural analyses and multi-marker barcoding, in conjunction with molecular species delineation analyses (ABGD, haplotype networks, GMYC, and bPTP). In total 132 specimens of Caecum and Meioceras collected during several sampling trips to Central America were analyzed and delineated into a minimum of 19 species to discuss putative synonyms, and supplement the original descriptions. Molecular phylogenetic analyses suggest Meioceras nitidum and M. cubitatum should be reclassified as Caecum, and the genus Meioceras might present a junior synonym of Caecum. Meiofaunal caecids morphologically resembling C. glabrum from the Northeast Atlantic are a complex of cryptic species with independent evolutionary origins, likely associated with multiple habitat shifts to the mesopsammic environment. Caecum invisibile Egger & Jörger, sp. nov. is formally described based on molecular diagnostic characters. This first integrative approach towards the taxonomy of Caecidae increases the known diversity, reveals the need for a reclassification of the genus Caecum and serves as a starting point for a barcoding library of the family, thereby enabling further reliable identifications of these taxonomically challenging microsnails in future studies.


Introduction
In the past fifteen years molecular barcoding and molecular species delineation have revolutionized the assessment of species diversity and traditional taxonomy, allowing for fast and reproducible species identification and delimitation, and adding to objectivity and reliability in species diagnoses (Leasi et al. 2013;Fontaneto et al. 2015;Scarpa et al. 2016;Martínez-Arce et al. 2020). Molecular data enables testing for morphologically cryptic species as well as phenotypic plasticity, and the evaluation of intraversus inter-specific variability (Jörger et al. 2012;Leasi et al. 2013Leasi et al. , 2016. Given the number of described species and 250 years of taxonomic practice that delimit species based largely on distinct morphologies, it is unsurprising that despite the success of modern molecular approaches, many clades of Metazoa have yet to have their morphological classification tested against molecular markers. Traditionally, the taxonomy of Gastropoda, one of the most species-rich and betterknown clades of invertebrates in the marine environment, is largely based on shell characteristics (Bouchet and Strong 2010). However, this approach is generally problematic as several studies have revealed species exhibiting phenotypic plasticity in shell form due to environmental factors or predation (Trussell 2000;Weigand et al. 2011), and uncovered cryptic species with the aid of molecular data (Haase et al. 2007;Puillandre et al. 2010;Jörger et al. 2012). Consequently, these studies question evolutionary hypotheses based on species delimited by shell characteristics alone and point to the need for an integrative approach using both molecular and morphological data in future research.
Members of the family Caecidae Gray, 1850 can be found in different marine habitats (e.g., among algae or corals) including the marine mesopsammon (i.e., the aqueous interstitial pore spaces of marine sediments). As adults they have uncoiled tubular shells that are likely an adaptation to their infaunal lifestyle (Swedmark 1968). In early descriptions zoologists associated Caecidae snails with tusk-shells (nowadays known as scaphopod molluscs) (see e.g., Montague 1803) or classified them among annelid tube worms (Brown 1827; see Pizzini et al. 2013 for a classificatory history). Even after Caecidae were settled among gastropods (Clark 1849), with current phylogenetic hypotheses placing them among caenogastropod Truncatelloidea (Criscione and Ponder 2013), their unusual tubular shells still posed challenge to taxonomists. Caecid larval shells (protoconch) are usually planspirally coiled with two whorls (Bandel 1996) scribed the caecid fauna in the Indo-Pacific based on microsculptural investigations of the shell (Pizzini et al. 2013;Vannozzi 2017Vannozzi , 2019, knowledge of caecid diversity in Central American waters is still limited to light-microscopic identification of shells for a large majority of described species.
In this study we present data on caecid diversity based on several recent collecting trips to Central America. We identified the collected Caecidae specimens based on traditional taxonomy and used additional microsculptural observations and molecular barcodes to reliably assign different growth stages to taxa. We applied an integrative experimental approach including multi-marker barcoding and molecular species delineation analyses to test our morphology-based taxonomy, and to identify putative cryptic species.

Materials and methods
We collected and microscopically investigated a total of 132 individuals of meiofaunal caecid snails from five different sites in tropical Central America. Of 132 specimens, 67 were selected for further analyses (see Fig. 2 for sampling sites and Tables 1, 2 for details on material and sampling sites). Specimens were extracted from samples of coarse subtidal sands by resting them in buckets for at least 1-2 days to deplete oxygen and accumulate the meiofauna in the surface layer. The surface layer was skimmed off, and the snails extracted by a decantation technique after anesthetization with MgCl 2 -seawater solution using a sieve with a mesh size of 100 µm (Jörger et al. 2014). All specimens were documented alive and grouped into preliminary morphotypes based on light microscopic (LM) examination of shell characters in the field and fixed in 75-96% ethanol. Specimens provided by the Muséum national d'Histoire naturelle (MNHN) Paris had previously been removed from their shells in the field by the use of a microwave oven (Galindo et al. 2014), this method is advantageous and recommended over the destructive sampling described below, applied in the beginning of our survey.

Shell characteristics and microsculptural analyses
We documented the main taxonomic characters of the tubular shells ( Fig. 1), such as the morphology of aperture, septum, and mucro, and measured size and diameter of the shells. Initial species identification in the field was carefully revised in the laboratory, and specimens were assigned to species according to these shell characteristics. The microsculpture of the shell of one representative of each putative morphospecies was investigated via scanning electron microscopy (SEM), whenever a voucher was available (Table 1).
Microscopic debris on the shell was manually removed using an eyelash, and the shell rinsed in 96% ethanol. Specimens were dried by evaporation of the ethanol and transferred onto SEM stubs covered with self-adhesive carbon stickers. We used a sputter coater Polaron SC510 to coat the samples with gold in argon atmosphere. The shells were analyzed with a LEO 1430 VP SEM at a voltage of 15 kV.

DNA extraction, amplification, and sequencing
DNA was extracted from 121 of the 132 investigated specimens. The sputter-coated individuals previously investigated by SEM were crushed mechanically using pestles (Bergmeier et al. 2016); specimens investigated only by LM were also crushed if tissue was not already separated. Subsequently, DNA was extracted by the procedure of Knebelsberger and Stöger (2012) combining lysis with 2-mercaptoethanol in CTAB buffer, chloroform-isoamyl precipitation, and recovery using columns with silicamembrane (Nucleo Spin, Macherey-Nagel GmbH & Co. KG, Düren, Germany). The DNA was eluted twice with 25 µl aliquots of pre-heated elution buffer to gain high yield. DNA of specimens deposited to the USMN-Smithsonian Institution were extracted in the Laboratories for Analytical Biology, SI using the standard protocols of the Autogen Prep 956 Extractor (eluting with 100 µl Autogen R9 buffer). Three different markers were partially amplified by PCR: mitochondrial cytochrome oxidase subunit I (COI) and 16S rRNA gene, and nuclear 28S rRNA gene, using the standard PCR primers for gastropods (see Klussmann-Kolb et al. 2008 chemistry (PerkinElmer) and standard cycles (4 min denaturation at 96 °C, followed by 25 cycles of 10 sec at 96 °C, 5 sec at 50 °C and 4 min at 60 °C), and sequenced on an ABI 3730xl 96-well capillary sequencer. In total, 34%, 43% and 50% of the partial COI, 16S rRNA, and 28S rRNA gene sequences, respectively, were successfully amplified and sequenced. All sequences were edited in Geneious Prime (vers. 11.02011, Biomatters, Ltd., Auckland, New Zealand). Primer sequences were removed and base calls checked for misreads against their chromatogram. The sequences were then compared to sequences in the public database NCBI GenBank (http://ncbi.nlm.nih.gov/ genbank) by using the BLAST online web service to check for putative contamination. In total 29 COI, 40 16S rRNA and 43 28S rRNA gene sequences were deposited in NCBI GenBank (see Table 1 for accession and voucher numbers).

Phylogenetic analyses
Multiple sequence alignments of the 28S rRNA and COI genes were constructed using Mafft (vers. 7.419; Katoh et al. 2002;Nakamura et al. 2018)   for unambiguously aligned sites. Proposed exclusion sites were reviewed, adjusted, and subsequently removed (alignments before and after editing are deposited at https://doi. org/10.5281/zenodo.3613958). Sequences available from NCBI GenBank for in-group taxa (C. glabrum (Montagu, 1803) and C. glabellum (A. Adams, 1868)), as well as for out-group taxa were added (Table 3). Outgroups were assigned based on the recent molecular phylogenetic analyses by Golding (2014a, b) and Criscione and Ponder (2013). Two combined data sets were generated: (1) a concatenated alignment of all three marker genes and (2) a concatenated alignment comprising only the mitochondrial 16S rRNA gene, and COI. The data were combined into single matrices using P4 (Foster 2004). The combined data sets were then partitioned by gene and COI codon position.
Maximum likelihood (ML) and Bayesian inference (BI) were used to construct the phylogenetic tree from single genes and from combined and partitioned alignments. For each alignment jModelTest2 (vers. 2.1.10; Darriba et al. 2012) was run and the calculated likelihood scores weighted under the Akaike Information criterion (AICc) (Hurvich and Tsai 1989) which suggested GTR+I+G as the best fitting model. ML was performed using IQ-TREE (multicore vers. 1.6.7.1 for Linux 64-bit; Nguyen et al. 2014) with the GTR+G4+FO model (equivalent to GTR+G in RAxML vers. 8.2; Stamatakis 2014) with 300 bootstrap replicates. Bayesian MCMC analyses were performed using the program MrBayes (vers. v.3.2.6;Huelsenbeck and Ronquist 2001) with the same model. The Bayesian analyses were run in duplicates by default, with each run having four parallel Markov chains (MCMC) to estimate posterior probability support. Each chain was run for 5 million generations, sampling trees every 1000 th generation. Sampled trees were combined into a consensus tree after the first 1000 sampled trees (1000000 generations), considered as 'burn-in', were discarded. A general time-reversible model of nucleotide substitutions with a gamma-distribution of among-site rates (GTR+G) was used for the ML analyses. All trees were visualized

Species delimitation and characterization based on molecular data
Four different methods of species delineation were used with both the COI and 16S rRNA gene mitochondrial data sets. The Automatic Barcode Gap Discovery (ABGD) webserver was used to partition the data set into putative species based on the calculated gap between intra-and interspecific genetic differences (https://bioinfo.mnhn.fr/abi/ public/abgd/abgdweb.html; Puillandre et al. 2012). J ModelTest2 (vers. 2.1.10; Darriba et al. 2012) was applied to the uncorrected COI and 16S rRNA gene alignments and the parameters were weighted under the corrected Akaike Information criterion (AICc) (Hurvich and Tsai 1989). For both alignments the Jukes-Cantor (JC69) as well as Kimura (K80) model showed to be within the 100% confidence interval however, K80 had slightly higher likelihood scores. Both models were applied with the default settings (TS/TV = 2.0, relative gap width = 1.5, Pmin = 0.001, and Pmax = 0.10) on the uncorrected COI and 16S rRNA gene alignments.
To evaluate haplotype connectivity, we generated haplotype networks based on the COI as well as the 16S rRNA gene sequence alignment using the software TCS (vers. 1.21; Clement et al. 2000) using the standard 95% parsimony setting. Ambiguous sites in both alignments were removed to prevent the creation of artificial haplotypes.
The bPTP web server (https://species.h-its.org/) was used to conduct the Bayesian implementation of the PTP model for species delimitation (Zhang et al. 2013) on the optimal ML trees of the individual and the combined COI and 16S rDNA datasets. We applied the default settings with 100000 generations, thinning for each 100 th sample with a burn-in of 10% and checked for convergence of the MCMC chains of each run. Posterior probability (PP) support values above 0.95 were considered as strong support.
For the General Mixed Yule-Coalescent model (GMYC) (Pons et al. 2006), ultrametric trees from the COI, 16S rRNA gene, and combined COI and 16S rRNA gene data were obtained using a time calibrated Bayesian evolutionary analysis in Beast (vers. 1.7.4;Drummond and Rambaut 2007). For the tree prior, we used a Yule process and two fossil records, Caecum cooperi and Caecum imbricatum [2.58-1.80 myr] (Mansfield 1930;Cooke 1936;Ward and Blackwelder 1987) and the in-group Caecum [50-55 myr] (Goedert and Raines 2016) with a lognormal distribution (logL). The analysis was run with the GTR substitution model and under a strict clock assumption. The analysis was started from a random tree and two Markov chains run for 10 000 000 generations with a sampling frequency of 1000. Convergence of the chains was checked in Tracer (vers. 1.7.4.; Rambaut et al. 2018) and effective sampling sizes (ESS) were confirmed as > 200 for all values (Rambaut et al. 2018). The first 10% of sampled trees were removed as burn-in and the trees were combined in TreeAnnotator (vers. 1.7.4.; Drummond et al. 2012) using the maximum clade credibility option and mean node height. The ultrametric trees were uploaded to the web server (https:// species.h-its.org/gmyc) for single, as well as, multiple threshold GMYC analyses.
The software QUIDDICH (vers. 1.0.0; Kühn and Haase 2019) was used to identify the diagnostic molecular characters of morphologically cryptic species. We extracted diagnostic characters of type 1 (i.e., characters, which distinguish each individual of the investigated species from other caecids with a fixed character state in the investigated species) and of type 2 (i.e., characters, which distinguish each individual of the investigated species from all other caecids, but vary also within the investigated species) from the COI, 16S rRNA gene and 28S rRNA gene alignments of the same dataset also used for the species delineation and phylogenetic analyses.

Molecular phylogeny and primary species hypothesis
In our phylogenetic analyses Caecidae form a well-supported clade (1.0 PP, 99% BS; Fig. 3). The two established genera Caecum and Meioceras, however, are not recovered as reciprocally monophyletic but instead species of Meioceras group among Caecum species in different parts of the tree (Fig. 3, taxa highlighted in yellow): M. nitidum sister to C. heptagonum (0.96 PP), and M. cubitatum sister to C. cf. semilaeve (no statistical support). The phylogeny groups the Caecidae into 21 clades which show moderate to high support values ranging from 0.95 PP/85% BS to full support (Fig. 3). Other clades are only statistically supported by one analysis (C. regulare, 92% BS) or do not have statistical support (C. donmoorei). The sister group relationships of C. pulchellum and C. regulare (1.0 PP, 95% BS), and C. cooperi and C. imbricatum (1.0 PP, 100% BS) are well supported; otherwise, deeper nodes and higher-level relationships among clades are not supported. In agreement with the molecular data, C. pulchellum and C. regulare as well as C. cooperi and C. imbricatum show morphological similarities in shell ornamentation and microsculpture. Inconspicuous specimens with smooth shells and few characters that were morphologically ascribable to C. glabrum or the American Pa-cific look-alikes like C. glabriforme are polyphyletic and form four lineages separated by branches of comparable length to morphologically distinct species (Fig. 3, highlighted in blue). These lineages are distinct from C. glabrum from the North Atlantic (Table 3) included in the analyses, indicating the presence of morphologically cryptic species in this 'C. glabrum species complex'.

Molecular species delineation
The methods that were used for species delineation are largely congruent with regard to the assignment of taxa to molecular operational taxonomic units (MOTUs), however individual analyses deviate and evidently differences occur due to incomplete sampling of one of the markers (Fig. 4, Table 1). Both PTP/ bPTP and GMYC (single threshold) delimit 21 MOTUs for the concatenated dataset of COI and 16S rRNA genes (excluding the species whose sequences were retrieved from NCBI GenBank, i.e. North Atlantic C. glabrum and Japanese C. glabellum). These results are in concordance with the preliminary species hypotheses based on morphological investigation and the molecular phylogenetic tree (Fig. 3) with the exception of additional splits of C. debile and C. regulare into two distinct MOTUs each. Caecum cf. teres resulted in a single species for 16S rDNA alone, and the Bayesian implementation of bPTP split M. nitidum into two separate species based on the 16S rRNA genes (however, support value for the split is 0.501%). The multiple threshold analyses in GMYC additionally splits C. invisibile sp. nov. of the 'C. glabrum complex' into two MOTUs, as does TCS but into differing entities. In analyses of individual datasets (numbers not directly comparable due to missing data) ABGD identified 15 MOTUs in our 16S rRNA gene dataset (Fig. 4), while the COI dataset resulted in a hypothesis of 10 MOTUs independent of the application of the JC69 or the K80 model. In comparison to the other methods, TCS appears to oversplit MOTUs (see e.g., TCS analyses of the COI of C. donmoorei in Fig. 4). The algorithm of this haplotype-network software splits the 16S rDNA dataset into 19 independent haplotype networks, while it recovered 13 networks for the COI dataset (Fig. 4). Additionally, TCS also splits MOTU II of the 'C. glabrum-like complex' into two networks and C. donmoorei into three independent networks based on 16S rRNA sequence data. Haplotype networks divided C. cf teres and C. cf. strangulatum into two unconnected networks. However, the split is not congruent with the two monophyletic sister populations of the species tree (Fig. 4). In summary, we consider only splits relevant, which are supported by at least two different analyses or markers, singular deviating signal might either resemble errors in analyses or might be informative in population analyses (for more details see remarks in Systematics section). Based on the molecular phylogeny, specimens identified as Meioceras nitidum and M. cubitatum both group among Caecum species and should therefore be transferred to this genus. However, considering that only one M. nitidium is statistically supported, in the interest of taxonomic stability this finding is pending further molecular studies, once additional material is available, preferably including material from the type localities.
Remarks. Our molecular phylogenetic results delimited M. cubitatum as a separate species, despite similarities to M. nitidum in its bulbous shell shape and pattern. Surprisingly, our molecular analyses do not retrieve these morphologically similar Meioceras species as a monophyletic entity but suggest independent origin within Caecum. Morphological differences towards M. nitidum (characterized above) are a more slender shell with more pronounced curvature towards the anterior end and the opaque color of the present individual. We assigned the specimen to Meioceras cubitatum sensu de

Cryptic lineages revealed in molecular analyses
Twenty-four specimens from Central American waters are smooth and glossy without ornamentation except for occasional growth lines (i.e., possess few shell characteristics), but vary in adult shell length between 0.7 and 2.5 mm (Figs 3, 5F-L, O-R). Morphologically, these specimens all closely resemble Caecum glabrum (Montagu, 1803) which is one of the best-known species of caecids, and abundant in the northern Atlantic (Montagu 1803;Wood and Harmer 1848;Götze 1938;Chambers 2009). Caecum glabrum was originally described from Biddlesford Bay and Barnstable, Devon, England, the included sequences from GenBank (see Table 3) originates from specimens collected in Norway, but own unpublished data from Roscoff, northern France, supports the wide distribution range of C. glabrum along European coastlines based on molecular data. We refer to cryptic species with simple shells lacking characteristic features as 'Caecum glabrum-like' species complex. In previous works, specimens similar to C. glabrum have also been described from the Pacific (C. glabellum as Brochina glabella A. Adams, 1868 from Akashi, Japan, C. glabriforme Carpenter, Molecular diagnostic characters. see Table 4. Morphological description. All investigated specimens were very similar in appearance, with little or no variation in shell morphology. Shell completely translucent. Length 0.8 mm long, width 0.2 mm (holotype, Fig. 5O). Tube regularly curved, shape equal in width but bears prominent edge at transition to septum, edge with smaller diameter. Septum round and blistered lacking a distinct mucro. Septum slightly inclining towards the left, dorsal side in holotype with slight variation between the specimens. Aperture equally wide as tube with straight edge. Sculpture appears smooth, only with faint growth lines (Fig. 5R). Whitish translucent body visible through translucent shell. Operculum translucent, slightly tinted yellowish. Radula formula shows taenioglossate pattern 2.1.1.1.2. with very small central rhachidian tooth. Large lateral teeth oriented towards the rhachidian tooth. Marginal teeth finer, outer marginal teeth are scoop-like curved. All the specimens investigated are adults based on the cylindrical shape of the tube and the shape of the aperture showing a reflected lip without cutting edge, which is normally present in immature specimens.
Etymology. The Latin adjective invisibile (invisible, unable to be seen) refers to the minute size of specimens, the translucent color of its shell, its hidden lifestyle between sand grains, and its taxonomic crypsis. Remarks. Caecum invisibile sp. nov. is described as a new species based on molecular diagnostic characters, which show it as distinct from the European C. glabrum (Fig. 5M), as well as the morphologically similar C. corrugulatum (Fig. 5F, G) from the Central American Pacific and C. glabellum from Japan (Fig. 5N). Morphological characterization. Shell size 1.3 mm long, 0.3 mm wide. Translucent, with whitish body. Tube regularly curved and equal width. Septum hemispherical (Fig. 5K). Aperture straight, with lip indicating an adult specimen. Operculum brownish. No sculpture or microsculpture diagnostic features (compare Fig. 5L).
Remarks. MOTU I is highly similar to the European C. glabrum (Fig. 5M) and Caecum invisibile sp. nov. (Fig. 5O-R). However, MOTU I shows some small morphological differences such as a bigger shell size and a tiny rim at the aperture (Fig. 5L) which is absent in C. glabrum. The septum is further completely round and blistered (Fig. 5K), whereas the one of Caecum invisibile sp. nov. slightly inclines (Fig. 5Q). MOTU I is based on a singleton and an incomplete molecular dataset, lacking COI sequence data. Additional material from the same locality is necessary to justify proper species description in future research. Morphological characterization. Shell size unknown. Translucent, with translucent body. Tube regularly curved, slightly increasing in diameter towards aperture. Septum round, slightly flattened (Fig. 5H). Aperture straight. No sculpture visible, microsculptural data missing.
Remarks. MOTU II is based on the molecular data of three specimens; however, we unfortunately lack SEM scans and thus microsculptural data of the shell and light microscopic images are only available for one specimen (Fig. 5H). This specimen is a juvenile, and due to uncertainty with regards to adult ornamentation of the shell, and its possible identity with an already described species, we refrain from providing a formal description based on the available material only. Shell morphology. In juvenile specimens, shell fragile, translucent brownish color. Tube doubles diameter towards aperture, with a moderate curvature in anterior half, increasing distally in curvature. Septum level beneath cutting plane, slightly rising towards mucro (Fig. 6D). Mucro slender finger-like shape (Fig. 6D). Aperture fragile and partly broken. Shell sculptured by seven longitudinal ridges with transverse ribs crossing, knobs at intersections, ridges less prominent towards posterior. Microsculpture of fine rugose longitudinal stripes, noticeably increasing in width on transversal rings in comparison to interspaces (Fig. 6E).

Adding barcodes to known Central American Caecidae
Remarks. Due to the characteristics of the heptagonal tube with the transversal rings, considered unique among caecids (Lightfoot 1993a), the investigated specimen could be unambiguously assigned to C. heptagonum. However, illustrations of C. heptagonum indicate a very thick shell with distinct differentiated aperture with inner bulge forming a round opening instead of the outer polygonal shape (Keen 1974;Pizzini et al. 1998: 142, figs 1-13) including an inner bulge in the aperture, forming a round opening instead of the outer polygonal shape which is absent in the rather thin and fragile investigated specimens. As our samples only comprised juvenile specimen, however, we can attribute this variation to the unfinished shell state.
Remarks. See remarks on C. cooperi. " after paragraph on shell morphology of C. imbricatum.
Remarks. Our molecular species delimitation separates C. cooperi and C. imbricatum into two independent evolving sister species (see Figs 3,4). This is supported by (minor) morphological differences such as a finer but more pronounced bead-like ornamentation in C. cooperi in comparison to flattened squarish and less frequent longitudinal pattern. The included barcodes and distinguishing diagnostic features should help to overcome previous taxonomic uncertainty suggestive of synonymy (see C. imbricatum sensu Moore (1972: 888, fig. 6)). The putative synonymy of  Moore, 1969 andC. imbricatum (compare C. imbricatum sensu Tunnell et al. (2010: 144) = C. insularum sensu Moore (1970: 370, fig. 1A, B) needs to be tested by molecular data, ideally based on material recollected from the type localities.
Remarks. The present specimens were assigned to C. debile based on the characteristic microsculpture (see Absalão and Gomes 2001). Morphologically, C. debile might present a synonym of C. infimum, de Folin, 1867 (de Folin andPérier 1867: 26, pl .3, fig. 2) but, in awareness of cryptic species we refrain from synonymizing until C. infimum from the type locality is available for molecular analyses. Some species delineation analyses, separate C. debile into two independent species (Fig. 4), which might indicate a putative speciation, but more data on the genetic variability is needed to exclude the presence of an artefact in analyses.
Remarks. Caecum striatum was identified based on a comparison with the material collected in the sampling region and dedicated as lectotypes by Absalão and Gomes (2001). Their microsculptural description of the type material does correspond to the longitudinal striation of our investigated specimen. Furthermore, shape, mucro, and the noticeably sharp aperture are identical to our specimen. A comparison with a specimen of C. striatum pictured by Pastorino and Chiesa (2014: figs 10-16), also shows the same fine-lined microsculpture as our specimen. Type specimens of three highly similar species, namely C. johnsoni Winkley, 1809, C. antillarum Carpenter, 1858 and C. strigosum de Folin, 1867, are described from Central American waters. Differences can be compared in the reinvestigation of Absalão and Gomes (2001: 20, figs 39-41, 12, figs 11, 12 and figs 7, 8 respectively).
Remarks. The specimen corresponds to C. clathratum, which differs from other ribbed Caecum species by its exceptional size, golden color and lack of microsculpture (compare with Lightfoot 1993a: 15, fig. 1 and a syntype collected by Carpenter available through the online catalogue of the Natural History Museum London catalogue number 1857.6.4.1528). However, the specimen is described and known only from the Eastern Pacific. Our herein investigated specimen from the Atlantic might thus present a (morphologically cryptic) sister species new to science, which potentially originated when populations were separated via the formation of the Isthmus of Panama. But molecular data of specimens collected from the Eastern Pacific is required to confirm the molecular identity or justify the description of a new species.
Remarks. We identified the specimens as C. regulare by referring to the drawings of Carpenter's original description (Carpenter 1857;1858-1859, the syntype material from the Natural History Museum, London, UK accessed through the online catalogue (catalogue numbers 1858.12.9.19, 1858.12.9.20, 1858.12.9.21) and the redescription of Moore (1972): 888, fig. 7. The conspicuous widening of the shell very close to the aperture in our specimen investigated can be interpreted as a character of a sub-adult growth stage (Bandel 1996), thus not contradicting the assignment to C. regulare. A literature survey suggests at least one putative synonym of C. regulare, resp. Caecum planum, de Folin, 1874 (de Folin andPérier 1875: 277, t. 2, pl.10, figs 8, 9). However, the geographical distribution differs (C. regulare was originally described from the Caribbean and C. planum from Brazil), and there are no evident diagnostic differences to C. regulare. Hence, molecular data is needed for clarification. Further data also is needed for the complementary gene sequences (16S rRNA and COI) for the two investigated specimens. We consequently attribute the split of our two investigated specimens into distinct molecular species to missing data in our analyses.
Remarks. The collected specimen USNM 1618844 closely resembles the description of C. donmoorei from the Virgin Islands (Mitchell-Tapping 1979), however microsculptural data for comparison is missing. Our molecular data suggests the species identity among specimens with less separated, strongly flattened rings and almost vanishing interspaces (e.g., in juvenile USNM 1618857). The same has been observed for the confusingly similar species C. quadratum, Carpenter (1855Carpenter ( -1857). It can also exhibit a considerable variety of shell morphologies (Lightfoot 1993a), however it origins from the Eastern Pacific. And a second highly similar spe- Shell morphology. Thin, fragile shell. Color whitish translucent. Length varies from 1.2 to 1.5 mm. Tube elongated, uniformly cylindrical, narrowing towards posterior (Fig. 7M, N). Septum clearly set off from edge of tube with large, triangular mucro with rounded tip (Fig. 7Q). Aperture round with sharp and thin rim. Sculpture appears smooth using light microscopy except for numerous fine horizontal growth lines. Microsculpture composed of numerous narrow longitudinal stripes consisting of serial fine indentations (Fig. 7S). Stripes of indentations are slightly shifted, when intersecting a growth line.
Remarks. Our material from Pacific Panama closely resembles Caecum teres (lectotype, NHMUK catalog number 1857.6.4.1550). However, all investigated specimens are juveniles in different growth-stages and identification remains therefore to be confirmed when adult specimens are available for molecular analyses. Shell morphology. Shell very thin, delicate and highly translucent, glossy (Fig. 7U). Size 1.0 mm long, 0.5 mm wide. Tube gradually narrowing towards posterior end and evenly curved. Septum blistered, entirely below posterior tube end (Fig. 7W). Posterior end fringed and in specimen ZSM-Mol-20200034 still connected partly with mucro indicating a recent shedding of transitional septum. Mucro with elongated, rounded tip, only slightly extending from tube (Fig. 7W). Aperture bordered by very tiny sharp lip, otherwise fragile (Fig. 7X). Shell surface smooth, no sculpture visible apart from regular growth lines. Shell covered by organic layer (periostracum).
Remarks. The examined shells all belong to juveniles due to their fragile character and the unfinished aperture. Therefore, it will be critical to reassess these observations based on mature shell structures, as sculpturing is known to be variable during development (see e.g., C. metamorphosicum S. Lima, Santos & Absalão, 2013in Lima et al. 2013. The specimens investigated here build and shed transitional septa as described by Pizzini et al. (1998). Specimens that show a similar mucro are Caecum lineicinctum de Folin, 1880 (compare Absalão and Gomes 2001: 10, figs 1, 2), C. liratocinctum Carpenter, 1857; however, both occur in the Western Atlantic (de Folin 1880; Moore 1972;Lightfoot 1992a;Absalão and Gomes 2001). Caecum semilaeve is a species described as similar to C. liratocinctum (Carpenter 1855(Carpenter -1857 and its type locality is Mazatlán, Mexico, Eastern Pacific, thus with geographic proximity to the localities of our investigated specimens (Achotines, Panama, eastern Pacific). We therefore assign our material to C. semilaeve. However, identification remains uncertain without having observed the manifestations of the shell sculpture as described for C. semilaeve in later developmental stages (compare syntypes C. elongatum var. semilaeve NHMUK 1857.6.4.1526).

Taxonomic consequences for Caecidae and the fate of Meioceras
The Caecidae are currently classified in ten genera (MolluscaBase 2019) of which two, Caecum and Meioceras, can be found in the Central American region. We investigated two species classified as Meioceras and 15 Caecum species, including one species new to science (C. invisibile sp. nov.), and two candidate species (MOTU I and II). Three individuals that were originally assigned to Meioceras are resolved among species of the genus Caecum in our molecular phylogenetic analyses and, moreover, have independent evolutionary origins within Caecum (Fig. 3). Our data confirms the existence of at least two valid Meioceras species (i.e., M. nitidum and M. cubitatum), which however, based on our data should be reassigned genus Caecum. For taxonomic stability, we refrain to reallocate these species at present, until the molecular sampling can be expanded and further data is available supporting our initial results. Unfortunately, we lack material of M. cornucopiae (the type species by subsequent designation) and of a putative fourth species, M. tumidissimum, both described from Brazil (de Folin and Périer 1869). These taxa are needed to settle the debate on the number of species and to clarify the validity of the genus Meioceras. Our findings indicate, however, that the more bulbous shell in Meioceras when compared to a more tube-like shell in Caecum might not justify generic subdivision. Moreover, the diagnostic spiral growth pattern in the larval and juvenile shell of Meioceras (Bandel 1996) might not be used for unambiguous discrimination as our phylogeny indicates that such patterns have evolved independently at least two times within the genus Caecum. The bulbous adult shell with an oblique constricted aperture was thought to develop from the preceding ontogenesis of a helicoidal shell section, observable at the beginning of the second growth stages (De Folin 1880; Absalão and Pizzini 2002). This diagnosis has been problematic because in the past other species (Absalão and Pizzini 2002) that also express a similar shell-shape had been classified as Caecum due to a lack of observations of the juvenile stadia or the lack of the afore-mentioned growth pattern (e.g., Caecum ryssotitum, Bandel 1996). By contrast, species with typical tube-shaped Caecum-shells are known to also have curved growth axes (e.g., C. antillarum and C. japonicum referred to as C. glabellum in Bandel 1996: 65, figs 8, 9). Differences between these growth patterns and those of Meioceras seem to be negligible. Thus, it remains to be tested whether the remaining two Meioceras species form a monophylum separate form Caecum and whether alternative diagnostic morphological or molecular characters can be found to justify the generic subdivision of Western Atlantic Caecidae. Alternatively, such studies may confirm that Meioceras is a junior synonym of Caecum. The present study might also have consequences for the recently established genus Mauroceras, which unites Indo-Pacific Caecidae formerly classified as Meioceras (Vannozzi 2019). But, in contrast to Caecum and Western Atlantic Meioceras, which cannot be clearly separated based on variable growth patterns, Mauroceras is diagnosed by a planorbid protoconch with a clear sinusigera, which at present justifies its generic status.

Phylogenetic interpretation of shell morphologies and general insights for shell-based taxonomy
The taxonomy of Central American Caecidae has been based on macroscopic shell characters and, consequently, type-species are often poorly defined, and has made the established taxonomy prone to multiple descriptions of synonyms and the establishment of ambiguous species-complexes that are typical for many clades of micromolluscs (Golding 2014b). Modern microsculptural analyses have greatly increased the reliability of shell-based taxonomy and the availability of diagnostic characters in the otherwise largely featureless caecid shells (Pizzini et al. 2013;Vannozzi 2017). However, distinct shells based on coarser diagnostic features can have a similar microsculpture (Vannozzi 2017), suggesting that shell microsculpture should be co-evaluated with traditional diagnostic features and, indeed, that it might be especially valuable to discriminate closely related species. In Central American Caecidae the presence of a series of morphologically highly-similar ribbed taxa (i.e., C. compactum Carpenter, 1857, C. quadratum, C. clathratum, C. gurgulio Carpenter, 1858, C. pulchellum, C. regulare, Caecum planum de Folin, 1874 with controversial species status and inconsistent synonymization (Moore 1972) are especially problematic. Here we report SEM-based shell microsculpture that can distinguish taxa and justify the independent species status of C. regulare and C. pulchellum (compare fusiform lobes (Fig. 7C) with fine longitudinal lamellae (Fig. 7A). However, C. clathratum does not possess a unique microsculpture and we are lacking SEM data for our investigated specimen of C. donmoorei. Nevertheless, molecular species delineation analyses confirm the existence of four genetically distinct species among those Central American ribbed caecids (i.e., C. pulchellum, C. regulare, C. donmoorei and C. cf. clathratum), highlighting the value of complementary molecular analyses to detect possible synonyms or confirm the validity of existing species in taxonomically problematic species complexes.
The different growth stages of caecid development present an additional problem for taxonomic circumscription, which cannot be overcome easily by microsculptural analyses because, the shape and some patterns of ornamentation appear late in development. This often results in the incorrect assignment of different growth stages even at the generic level (Absalão and Pizzini 2002). In consequence, it requires time consuming comparisons of hundreds of shells for reliable species description and identification (Lightfoot 1992a(Lightfoot , 1993a, unfeasible in modern times of taxonomic impediment. The molecular analyses presented in this study show that barcoding markers (i.e., partial mitochondrial COI and 16S rRNA genes) are a valuable tool to address the challenges of caecid taxonomy and that molecular species delineation analyses can reliably identify groups of closely related specimens, therewith providing objective data on intraspecific variability of shell characters. Above all, they enable an unambiguous assignment of juvenile forms in different growth stages to their fully developed adult morphologies (see e.g., C. heptagonum in Fig. 5G, C. pulchellum and C. cf. teres in Fig. 7A, D). Based on a purely morphological approach, these juveniles would have remained unidentified and unaccounted for in biodiversity data, and their contribution to caecid diversity would have been lost. However, in some cases, juveniles could not be matched to their adult counterparts using molecular data since we had no adult animals in our sample. These taxa identified by the molecular data could not be named (e.g., Caecum sp. MOTU II). These examples highlight, how the successful identification of juveniles lacking morphological diagnostic features by means of their genetic fingerprints requires an extensive barcode library of Central American Caecidae as a taxonomic reference. The barcodes of the morphospecies investigated here are the first contribution to such a reference library that can help to provide a baseline and enhance future identification. In general, the poor taxonomic coverage of gastropods and marine invertebrates in public molecular databases such as NCBI GenBank has been identified as a major obstacle to making effective use of molecular barcoding approaches (e.g., to assign spawn to adult specimens; Puillandre et al. 2009). Thus, it is hoped that in the future the scientific community will be able to invest more of its financial and personnel capacities in integrative faunistic approaches that strengthen fundamental biodiversity research.
In biodiversity assessment and conservation biology, molecular species delineation has also demonstrated its potential for identifying cryptic species (Bickford et al. 2007;Jörger et al. 2012;Lemer et al. 2014;Leasi et al. 2016). In revealing cryptic taxa, our study indicates that the species diversity of caecids may have been underestimated until now. Unsurprisingly, the cryptic species, which we identified, are those of particularly small, feature-poor, caecids with few diagnostic characters (see Fig. 5C-F). Indeed, our analyses suggest that meiofaunal character-poor caecids (assigned to the 'Caecum glabrum-like' species complex) have evolved several times independently from the larger ornamented caecids in the Central American region. The same may have happened in the northern Atlantic C. glabrum and Northwest Pacific C. glabellum Adams, 1868 from Japan. The evolution of a tubular shell marks the origin of Caecidae and likely correlates with a transition to an infaunal lifestyle (e.g., among corals and coral rubble or algae; Bandel 1996). However, interstitial habitats are very variable, differing with regards to the available space between the sand grains which influences the mobility, light intensity and therefore visibility and protection from predators.
In the 'Caecum glabrum-like' microsnails, the morphological similarity among taxa (i.e., minute, slim shell, lack of ornamentation and coloration) likely correlates with a habitat shift into the mesopsammon and the consequent habitat restrictions of this special interstitial environment. 'Regressive evolution' leading to simplified and highly adapted body plans are typical for the mesopsammon (Swedmark 1968) and consequently the associated meiofauna is prone to cryptic speciation (Jörger et al. 2012;Meyer-Wachsmuth et al. 2014;Leasi et al. 2016).

Conclusions
Our study of Central American Caecidae shows that traditional taxonomic shell characters cannot sufficiently describe the diversity of these microsnails. Microsculptural investigations add valuable additional information for correct taxonomic assignment, species delineation, and the evaluation of gross shell morphological variation within and among species. However, its effectiveness in allocating juvenile growth stages or morphologically rather cryptic species with few diagnostic shell characters into the classificatory system remains limited. This limitation in morphology-based approaches was overcome by integrating genetic barcoding data and molecular species delineation which revealed a complex of cryptic lineages that were potentially associated with a habitat shift from an epibenthic to (temporary) mesopsammic lifestyle among the interstices of sand grains and shell hash. Integrative biodiversity assessments help contribute to a barcoding library of genetic fingerprints of the targeted fauna which enable rapid identification of new samples and is linked to the existing taxonomic history by morphological identification of the voucher specimens. Thus, beyond documenting the shell in microstructural detail, whenever possible a shell voucher should remain intact available for future investigation when novel methods approach. Nevertheless, the vast accumulation of potential synonyms and old names in gastropod taxonomy is problematic, and species need to be taxonomically revised prior to establishing names for newly discovered species. Re-collecting at type localities might not always be feasible for each species, especially when revising large groups with many described species. Additionally, it bears the risk of false identification when cryptic species co-occur at small geographical ranges. However, genetic barcodes have been generated successfully from old mollusk samples in natural history collections -wet material (Jaksch et al. 2016) and dried shells (Der Sarkissian et al. 2017) alike -and hopefully advances in genetic methodology will soon provide cost-efficient and reliable workflows to also adapt them to microsnails as a complement towards ongoing biodiversity studies.