Gyratrix hermaphroditus, Ehrenberg, 1831, Ehrenberg, 1831

Tessens, Bart, Monnens, Marlies, Backeljau, Thierry, Jordaens, Kurt, Steenkiste, Niels Van, Breman, Floris C., Smeets, Karen & Artois, Tom, 2021, Is ‘ everything everywhere’? Unprecedented cryptic diversity in the cosmopolitan flatworm Gyratrix hermaphroditus, Zoologica Scripta 50 (6), pp. 837-851 : 842-843

3 | R E S U LT S

3.1 | Phylogeny and species delimitation

The 3,906 bp long concatenated alignment (18S: 1,781 bp; 5.8S: 105 bp; ITS2: 354 bp; 28S: 1,666 bp) contains 1,346 (34.5%) variable sites (18S: 316; 5.8S: 34; ITS2: 257; 28S: 751). As such, the concatenated sequences of 401 specimens of G. hermaphroditus included here yield 229 different haplotypes. The mean/maximum uncorrected p-distances among haplotypes are very high (18S: 0.028 / 0.053; 5.8S: 0.045 / 0.126; ITS2: 0.159 / 0.306; 28S: 0.065 / 0.107) for what are generally considered slow-evolving genes (18S, 5.8S) ( Hillis & Dixon, 1991). BI and ML tree topologies are nearly identical (Figure 1a) and reveal deep divergences and many well-supported clades.

The GMYC model supports the presence of cryptic species in G. hermaphroditus (likelihood ratio test, p <0.001). A multiple-threshold model did not fit the data significantly better than the single-threshold model (χ 2 = 8.83, p = 0.453). A total of 78 putative species consisting of 50 clusters and 28 singletons were identified with the GMYC method (confidence interval 72–82, Figure 1a, Table S1). All GMYC clusters are well supported (posterior probability/pp> 0.95, bs> 75), except for clusters C3 (not retrieved by BI, bs = 78), D4 (pp = 0.98, bs = 67), G1 (pp = 0.65, bs = 95) and I4 (pp = 0.99, bs = 50).

ABGD results suggest 62 putative species with 38 clusters and 24 singletons (Figure 1a, Table S1). All ABGD clusters are well supported (pp> 0.95, bs> 75), except for cluster A3 (pp = 0.65, not retrieved in ML analysis). All GMYC species correspond to or are nested within the ABGD species.

3.2 | Morphology and morphometrics

Based on subtle variations in the shape and relative length of the sclerotised structures of the male copulatory organ, 14 morphotypes were distinguished (Figure 1b) and related to the different clades in the phylogeny (clades A–O, Figure 1a).

The lengths of the stylet, sheath and stalk were significantly correlated (Table S3). Therefore, measurements of the sheath and stalk relative to the length of the stylet were used in subsequent analyses (Table S3). For morphotypes A, C, E, H, I, L and N, we were able to test whether morphometric data discriminate between putative GMYC species within each morphotype. DA clearly separated the GMYC clusters within each morphotype (Figure S1), and there was a low misclassification proportion (0 to 10.4%, Table S4). Even shallow GMYC clades (e.g. A1, A2, A3 and N1, N2, N3) were successfully discriminated by the morphometric data (Figure S1, Table S4).

3.3 | Biogeography

A total of 35 GMYC species (45%; 17 freshwater, 17 marine, 1 brackish) and 22 ABGD species (36%; 11 freshwater, 10 marine, 1 brackish), excluding those represented by a single specimen, were found at single sites. An additional 20 GMYC species (26%; 13 freshwater, 5 marine, 1 freshwater + marine, 1 freshwater + brackish) and 18 ABGD species (30%; 12 freshwater, 5 marine, 1 freshwater + marine) were found at locations separated by <100 km. Conversely, eight GMYC species (3 freshwater, 5 marine) have wide distributions within a single continent (> 500 km apart) and three GMYC species (1 freshwater, 2 marine) were found on at least two continents. ABGD revealed even more putative species with very wide distributions: eight ABGD species (3 freshwater, 5 marine) separated by> 500 km within a single continent and six ABGD species (4 freshwater, 2 marine) on more than one continent. Each of the six ABGD species with the widest distributions formed interconnected statistical parsimony networks at the 95% confidence level (Figure 2). In these networks, haplotypes were never shared between different geographic regions.

Many putative species occur sympatrically, i.e. at exactly the same site: 34/37% of freshwater and 59/57% of marine sites contain more than one GMYC/ABGD species. While most instances of sympatry pertain to species with differing copulatory organs, six (GMYC & ABGD) cases of identical morphotypes co-occurring were also observed. There were on average 1.7/1.8 GMYC/ABGD species per site, with a maximum number of 7/9 GMYC/ABGD species found at a single site (a beach near Alghero, Sardinia).

3.4 | Habitat

All putative species occur either exclusively in marine, brackish or freshwater habitats, except clade A3/A1 GMYC/ ABGD, which contains both freshwater and brackish water (= Baltic Sea) populations (Figures 1 and 3). No habitat type conforms to a single monophyletic group. Indeed, the brackish water taxa are distributed over three unrelated clades deeply nested in otherwise freshwater (node 4, Figure 1a) or marine (node 3, Figure 1a) clades.