Gint gubanensis, Kovarik, Lowe, Just, Awale, Elmi & Stahlavsky, 2018

Just, Pavel, Šťáhlavský, František, Kovařík, František & Štundlová, Jana, 2022, Tracking the trends of karyotype differentiation in the phylogenetic context of Gint, a scorpion genus endemic to the Horn of Africa (Scorpiones: Buthidae), Zoological Journal of the Linnean Society 196 (2), pp. 885-901 : 893

GINT GUBANENSIS

Karyotype of G. gubanensis comprises 2 n = 45 chromosomes, which decrease in length from 2.97% to 1.02% of DSL (Supporting Information, Fig. S2G ; Table S2). Overall, 21 bivalents and a single trivalent, composed of chromosomes 7, 33 and 45, are detected in post-pachytene nuclei ( Fig. 3I ; Supporting Information, Fig. S2G ). One pair of 18S rDNA loci is found on a terminal part of the heteromorphic chromosome pair (chromosomes 26 and 44). A single large 18S rDNA cluster is located on the overhanging part of chromosome 26 (the larger chromosome of the heteromorphic pair) ( Fig. 3J ; Supporting Information, Fig. S2G ).

MTDNA DIVERSITY AND PHYLOGENY

The sequences for both 16S and COI were obtained from 22 Gint individuals. In G. calviceps , only the 16S gene fragment was successfully sequenced. The final alignment with a total length of 963 bp (16S: 358 bp; COI: 605 bp) consisted of 765 conserved sites, 194 variable sites and 172 parsimony-informative sites.

Based on COI data, Gint exhibits genetic variation at both inter- and intraspecific levels, with the pairwise genetic distances ranging from 3.28% to 11.52% between species, and from 0.00% to 2.64% within species (Supporting Information, Tables S3, S4). The haplotype network reconstruction showed a presence of 15 COI sequence haplotypes in Gint (Supporting Information, ( Fig. S3 ; Table S3). Within the species studied, G. banfasae shows the highest within-location haplotype diversity (four haplotypes per locality), but with minor pairwise genetic differences (p -distance: ≤0.66%). In this species, all individuals of the same cytotype have distinct haplotypes. The haplotypes of cytotype I individuals (S1531 and S1534) are separated from one another by four mutational steps. Cytotype II (S1532) shares its haplotype with the cytotype III individual (S1530), which is one mutational step distant from the haplotype of the cytotype III specimen (S1533) (Supporting Information, Fig. S3 ). Gint dabakalo , comprising three haplotypes, shows the highest intraspecific genetic variation with a p -distance of 0.17–2.64%. In this species, the genetic distance between individuals of cytotype I and cytotype II from the same locality is higher than between each of them and a specimen of cytotype III from a distinct locality (Supporting Information, Fig. S3 ; Table S1). Gint maidensis , a species with a stable karyotype 2 n = 34, is represented by three haplotypes with a p -distance of 0.33–0.50%. Gintgaitako presentstwohaplotypes, where one haplogroup, formed by five specimens, is distant from the other by one mutational step (p -distance: 0.17). In G. amoudensis , individuals of cytotype II, cytotype III and cytotype IV share the same haplotype, whereas the specimen of cytotype I differs from this haplogroup by eight point mutations (p -distance: 1.49%).

Both BI and ML mtDNA analyses provide similar tree topologies and nodal supports for clades. Phylogenetic reconstruction depicts Gint as a monophyletic group ( Fig. 5 ). Most of the Gint species form well-supported clades [bootstrap support (bs) ≥ 83, posterior probability (PP) ≥ 0.98]. The monophyly of the G. banfasae specimens remains unresolved due to low support values. Gint maidensis forms an early-branching clade, which is sister to the clade comprising all of the remaining Gint species. This clade is further subdivided into two groups: (1) a clade in which G. gubanensis is a sister-species to G. amoudensis , and (2) a clade consisting of G. gaitako , G. calviceps , G. dabakalo and G. banfasae . In the latter, G. gaitako forms a sister-lineage to the clade comprising G. calviceps , G. dabakalo and G. banfasae . The mutual relationships between the species in this group remain unresolved due to low support values.

THE RECONSTRUCTION OF ANCESTRAL CHROMOSOME NUMBER AND POSITION OF 18S RDNA GENE CLUSTERS

Maximum parsimony analysis inferred 2 n = 32–34 as the ancestral chromosome number for the genus Gint ( Fig. 6A ). Two independent increases of 2 n were detected within Gint : (1) the clade containing all cytotypes of G. amoudensis and G. gubanensis , and (2) cytotype III within the G. dabakalo clade. When compared with the ancestral 2 n, a tendency of decreasing of 2 n from 30 to 18 chromosomes can be observed in G. gaitako , G. dabakalo and G. banfasae . Concerning the position of 18S rRNA gene clusters, maximum parsimony inferred their terminal position as the ancestral state for the genus Gint ( Fig. 6B ). Two independent changes of the ancestral terminal position to a subterminal position were identified in G. amoudensis and in the clade containing G. dabakalo and G. banfasae .

Figures

Figure 2. Post-pachytene cells of Gint species after Giemsa staining (A, C, E, G, I, K, M, O) and FISH with 18S rDNA (red signals) (B, D, F, H, J,L, N, P).A, B,G. banfasae cytotype I(2n=18 –7II + IV).C, D, G. banfasae cytotype II (2n= 18 – 6II +VI). E, F, G. banfasae cytotype III (2n = 19 – 5II + III + VI). G, H, G. dabakalo cytotype I (2n = 23 – 9II + V). I, J,G. dabakalo cytotype II (2n = 24 – 8II + III + V). K, L, G. dabakalo cytotype III (2n = 27 – 8II + 2III + V). M, N, G. gaitako (2n = 30 – 13II + IV). O, P, G. maidensis (2n = 34). Abbreviations: II, bivalent; III, trivalent; IV, quadrivalent; V, pentavalent; VI, hexavalent. Arrowheads indicate the position of 18S rDNA. Scale bar = 10 µm.

Figure 3. Post-pachytene cells of Gint amoudensis and G. gubanensis after Giemsa staining (A, C, E, G, I) and FISH with 18S rDNA (red signals) (B, D, F, H, J). A, B, G. amoudensis cytotype I (2n = 36 – 16II + IV). C, D, G. amoudensis cytotype II (2n = 35 – 14II + III + IV). E, F, G. amoudensis cytotype III (2n = 36 – 15II + VI). G, H, G. amoudensis cytotype IV (2n = 35 – 13II + III + VI). I, J, G.gubanensis (2n = 45 – 21II + III). Abbreviations: II, bivalent; III, trivalent; IV, quadrivalent; VI, hexavalent. Arrowheads indicate the position of 18S rDNA. Scale bar = 10 µm.

Figure 5. Bayesian tree of the Gint species based on the concatenated data set (16S and COI), complemented with summarized cytogenetic data of the species studied. Numbers above branches correspond to values for highly supported nodes as follows: Bayesian posterior probabilities (PP)> 0.95/maximum likelihood bootstrap> 70%. Specimen IDs depicted in bold indicate individuals based on chromosome counts determined for the corresponding Gint species in previous studies (see: Kovařík et al., 2013; Kovařík & Mazuch, 2015; Kovařík et al., 2018). Abbreviations: cyt, cytotype; 2n, diploid number of chromosomes; II, bivalent; III, trivalent; IV, quadrivalent; V, pentavalent; VI, hexavalent.

Figure 6. Ancestral state reconstruction analyses based on the Bayesian tree from this study, depicting: A, number of chromosomes; B, position of 18S rDNA. Results of maximum parsimony analysis reconstructing ancestral states for both cytogenetic characters are visualized by colours of the circles. Arrowheads indicate hypothetical inversion on 18S rDNAbearing chromosomes.