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Figure 2. Mitotype tree and distribution maps for 98 in Integrative taxonomy reveals cryptic diversity in North American Lasius ants, and an overlooked introduced species

Figure 2. Mitotype tree and distribution maps for 98 DNA-barcodes belonging to 7 mitotypes of the ant Lasius niger (blue, n = 70) and 15 mitotypes of L. ponderosae sp. nov. (red, n = 28). The red dashed line delimits the expected natural range of L. ponderosae sp. nov.53 Maps have been created using the free R-package "ggmap" v3.0.0 (https://github.com/dkahle/ggmap) in R v4.1.1. Map tiles by Stamen Design, under CC BY 3.0.

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Figure 3 in Integrative taxonomy reveals cryptic diversity in North American Lasius ants, and an overlooked introduced species

Figure 3. Frontal, lateral and dorsal view of the holotype worker (a–c), a paratype gyne (d–f) and a paratype male of Lasius ponderosae sp. nov. (g–i).

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Figure 1 in Integrative taxonomy reveals cryptic diversity in North American Lasius ants, and an overlooked introduced species

Figure 1. Molecular phylogeny of 26 Holarctic ant taxa belonging to the subgenus Lasius sensu Wilson (1955) and two outgroup taxa (L. pallitarsis and L. mixtus). The phylogeny was calculated under the coalescent model and incorporates data from 9 genes (mtDNA: COI, COII, 16S, nuDNA: Defensin, H3, LR, Wg, Top1 & 28S). Names of species native to the Nearctic are shown in red and those of species native to the Palearctic in blue. Node labels show posterior probability (Bayesian inference) followed by bootstrap support (Maximum likelihood). The scale bar indicates the length of 0.01 substitutions/site.

Fig. 9 in Disentangling the identity of Lebertia porosa Thor, 1900 using integrative taxonomy (Acari: Hydrachnidia)

Fig. 9. Lebertia (Pilolebertia) gibbosa Lundblad, 1926, holotype, ♂ (NHRS). A. Venter. B. Palp. C. I-L. D. II-L. E. III-L. F. IV-L. Scale bars = 100 µm.

Fig. 3. GenusLebertia Neuman, 1888 in Disentangling the identity of Lebertia porosa Thor, 1900 using integrative taxonomy (Acari: Hydrachnidia)

Fig. 3. GenusLebertia Neuman, 1888, positions and shorthand labelling for morphological measurements. A. Example of segment length and height measurements for legs and palps. B. Measurements across the coxal shield. C. The dimensions of the claw; the centre of curvature (c) was defined to be the point opposite of the smaller claw. D. Distances among the long setae on the third segment of the palp. Abbreviations: Ac = acetabula; Cx = Coxa; Gn = gnathosomal bay; L = length; mL = median length; W = height; II-L = second leg. Roman numerals refer to the order of legs starting anteriorly, Arabic numerals refer to segment number starting proximally, for Ac starting anteriorly.

Fig. 1 in Disentangling the identity of Lebertia porosa Thor, 1900 using integrative taxonomy (Acari: Hydrachnidia)

Fig. 1. Lebertia (Pilolebertia) porosa Thor, 1900 s. lat. A. Dorsal view. B. Ventral view showing acetabula. Photo: Reinhard Gerecke.

Fig. 2 in Disentangling the identity of Lebertia porosa Thor, 1900 using integrative taxonomy (Acari: Hydrachnidia)

Fig. 2. Neighbor Joining tree based on COI barcodes of Norwegian specimens in the Lebertia porosa aggr. using the Kimura 2-Parameter substitution model. Bootstrap support (1000 replicates) above 70% is shown on branches.

Fig. 6 in Disentangling the identity of Lebertia porosa Thor, 1900 using integrative taxonomy (Acari: Hydrachnidia)

Fig. 6. Lebertia (Pilolebertia) spp. Examples of observed differences in setation. A–B. High number of setae on segment five of legs three and four (III-L-5, IV-L-5) present in Lebertia aggr. spp. B and D. C. Gap in swimming setae on the fifth segment of the second leg (II-L-5) in Lebertia aggr. spp. A and B, and L. obscura Thor, 1900. D. Segment three of palp (P-3) with a double proximal long seta sometimes present in Lebertia aggr. spp. D. E–F. Segment five of the second leg (II-L-5), close-up: comparison of swimming setae with and without the large gap respectively.

Fig. 8 in Disentangling the identity of Lebertia porosa Thor, 1900 using integrative taxonomy (Acari: Hydrachnidia)

Fig. 8. Lebertia (Pilolebertia) obscura Thor, 1900, ♂ from the type locality. A. Coxal field. B. Palp. C. I-L. D. II-L. E. III-L. F. IV-L. Scale bars = 100 µm.

Fig. 7 in Disentangling the identity of Lebertia porosa Thor, 1900 using integrative taxonomy (Acari: Hydrachnidia)

Fig. 7. Lebertia (Pilolebertia) porosa Thor, 1900, ♂ from the type locality. A. Venter. B. Palp. C. I-L. D. II-L. E. III-L. F. IV-L. Scale bars = 100 µm.

Fig. 5 in Disentangling the identity of Lebertia porosa Thor, 1900 using integrative taxonomy (Acari: Hydrachnidia)

Fig. 5. Lebertia (Pilolebertia) spp. from Norway. TCS haplotype network of COI sequences constructed with PopART.

Fig. 4 in Disentangling the identity of Lebertia porosa Thor, 1900 using integrative taxonomy (Acari: Hydrachnidia)

Fig. 4. Lebertia (Pilolebertia) spp. in Norway. Maximum Likelihood tree from analysis of the concatenated dataset (COI, 18S, 28S) in RAxML-NG. Bootstrap support (500 replicates) above 50% on branches.

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Figure 2 in Integrated taxonomy supports the identification of some species of Phytoseiidae (Acari: Mesostigmata) from Georgia

Figure 2 Neighbour joining phylogenetic trees includingTranseius wainsteini from Georgia, Amblyseius swirskii, Amblyseius andersoniand Euseius stipulatus (as an outgroup) obtained with a – COI mtDNA, b – CytB mtDNA and c – 12S rRNA markers.

Fig. 6 in Polyclinum constellatum (Tunicata, Ascidiacea), an emerging non-indigenous species of the Mediterranean Sea: integrated taxonomy and the importance of reliable DNA barcode data Abstract

Fig. 6: ML phylogenetic tree of the genus Polyclinum (sequences abbreviation: Pln) based on COI nucleotide sequences (1560 aligned nucleotide sites; best-fit substitution model GTR+I+G; bootstrap on 100 replicates). Eudistoma and Pseudodistoma species were used as outgroups. The sequence list and species abbreviations are reported in Supplementary table S1. Black dots: bootstrap values ≥ 70 %; red: P. constellatum sequences; blue: P. indicum sequences; yellow background: our sequences.

Fig. 4 in Polyclinum constellatum (Tunicata, Ascidiacea), an emerging non-indigenous species of the Mediterranean Sea: integrated taxonomy and the importance of reliable DNA barcode data Abstract

Fig. 4: A, C) Colonies of Polyclinum constellatum with different colours photographed and collected in the Heraklion marina (Crete) (A: colony K11 and C: colony K12); B) Transversal section of the colonies, joined only at the surface layer (upper white arrow); D) Zooid extracted from the red-orange colony (K11), with magnification of the 6-lobed anus; E) Zooid extracted from the dark blue colony (K12) with magnification of the 6-lobed anus. Both K11 and K12 have the same COI haplotype (sequence AC number: MT873559).

Fig. 5 in Polyclinum constellatum (Tunicata, Ascidiacea), an emerging non-indigenous species of the Mediterranean Sea: integrated taxonomy and the importance of reliable DNA barcode data Abstract

Fig. 5: A) Larva of P. constellatum, showing the ocellus, four long narrow ampullae, three adhesive papillae and a group of a few small ventral vesicles (red arrow). am, ampullae; ap, adhesive papillae; oc, ocellus; B) Larva of P. constellatum, red arrow pointing out the calcite crystal in the middle of the body.

Fig. 2 in Polyclinum constellatum (Tunicata, Ascidiacea), an emerging non-indigenous species of the Mediterranean Sea: integrated taxonomy and the importance of reliable DNA barcode data Abstract

Fig. 2: A) Orange colony of Polyclinum constellatum from Taranto harbour (colony P1); B) Magnification of the oral (arrow pointing put the oral tentacles of different size) and cloacal aperture (asterisk); C) P. constellatum collected in Heraklion (colony K19) with zooids arranged in systems around the cloacal apertures; D) Section of the colony showing the zooids located only around the outer edge (arrow).

Fig. 3 in Polyclinum constellatum (Tunicata, Ascidiacea), an emerging non-indigenous species of the Mediterranean Sea: integrated taxonomy and the importance of reliable DNA barcode data Abstract

Fig. 3: A) Whole zooid of Polyclinum constellatum, showing a clear division into thorax, abdomen and post-abdomen with a long vascular stolon. ab, abdomen; pa, post-abdomen; th, thorax; vs, vascular stolon; B) Zooid with evident pharynx, rectum, anus and four embryos incubated in the atrial cavity. The funnel-shaped oesophagus, the smooth stomach and the twisted gut loop are visible in the abdomen. The post-abdomen shows the heart at its terminal end, as well as several rounded testicular follicles and the ovary, with the gonoducts running parallel to the rectum. an, anus; e, embryos; gd, gonoducts; gl, gut loop; oe, oesophagus; ov, ovary; h, heart; r, rectum; st, stomach; tf, testicular follicles; C) Magnification of the oral siphon with six pointed lobes (arrows) and six longitudinal muscle bands (indicated with numbers 1-6); D) Branchial sac with 18 rows of stigmata and narrow languets of the dorsal lamina (arrows); E) Magnification of the pharynx, with minute papillae (arrows) at the level of the transverse vessels; F) Magnification of the six-lobed anus (lobes indicated with numbers 1-6).

Fig. 1 in Polyclinum constellatum (Tunicata, Ascidiacea), an emerging non-indigenous species of the Mediterranean Sea: integrated taxonomy and the importance of reliable DNA barcode data Abstract

Fig. 1: Map of the Mediterranean Sea showing the literature records (black rhombuses) of P. constellatum and the new findings (red dots). performed in a final reaction volume of 25 μl contain- nus was reconstructed with the online software PHYML ing: 1X reaction buffer with 1 mM final concentration of v3.0 (http://www.atgc-montpellier.fr/phyml-sms/) (Guin- MgCl 2 (Takara Bio Inc.), 0.2 mM of each dNTP, 0.3 μM don & Gascuel, 2003), which also includes the automatof each primer and 1.25 Units of PrimeStar HS (Takara ic model selection algorithm SMS (Smart Model Selec- Bio Inc.). Amplification conditions were: 30 cycles with tion). The best-fit substitution model was selected using denaturation for 10 s at 98°C, annealing for 15 s at 46°C the Akaike Information Criterion (AIC). Bootstrap val- or 50°C, extension for 1 min 30 s at 72°C; a final elonga- ues, indicating node reliability, were based on 100 reption step of 5 min at 72°C. licates. The sequence dataset used for this phylogenetic PCRs with the DreamTaq polymerase were performed reconstruction is reported in Supplementary Table S1 and in a final volume of 25 μl containing: 1X reaction buffer was extracted from the phylogenetic dataset published in with 2 mM final concentration of MgCl 2 (Thermo Fish- Tabudravu et al. (2019). It includes representative species er Scientific), 0.2 mM of each dNTP, 0.4 μM of each of of the Polyclinidae family plus Eudistoma and Pseudodithe two primers, and 1.25 Units of DreamTaq polymerase stoma species chosen as outgroups for their morphologi- (Thermo Fisher Scientific). The amplification conditions cal similarities with Polyclinidae. were as follows: an initial denaturation for 3 min at 95°C, then 34 amplification cycles (denaturation for 30 s at 95°C; annealing for 30 s at 46-50°C; extension for 1 min Results 30 s at 72°C) followed by a final elongation step of 5 min at 72°C. Morphological analyses The obtained amplicons were purified with the DNA Clean&Concentrator kit (Zymo Research) and directly The colonies collected in Taranto harbour and Hersequenced according to the Sanger method by Microsynth aklion marina were all morphologically identified as P. AG (Switzerland). The sequence quality check, compar- constellatum based on the following features: colonies isons and alignment were carried out with Geneious ver. without sand in/outside, zooids arranged in systems, 5.5.7.2 (Kearse et al., 2012). The sequences obtained post-abdomen (without vascular stolon) shorter than the were deposited in the GenBank database (see Accession thorax and abdomen combined, pharynx with 16-18 rows numbers MT873559 and OL597608). For comparative of stigmata, more than 15 stigmata per row, and a 6-lobed analyses, homologous sequences of the genus Polycli- anus. These characteristics are in accordance with the key num were searched for in the non-redundant nucleotide of Polyclinum species edited by Kott (1963) and they are database (nr-nt db, on 21st September 2021) of the NCBI also reported in the description of the species made by (National Center for Biotechnology Information) by En- Van Name (1945). trez text search, and by BLASTn (Altschul et al., 1990) using our P. constellatum sequences as the query. Uncorrected pairwise distances were calculated with PAUP 4.0a (Swofford, 2002), while a Maximum Likelihood (ML) phylogenetic tree of the genus Polyclinum ge-