Buthids, C. L. Koch, 1837

Kovařík, František, Lowe, Graeme, Ranawana, Kithsiri B., Hoferek, David & Š, V. A., 2016, Scorpions of Sri Lanka (Scorpiones Buthidae, Chaerilidae, Scorpionidae) with description of four new species of the genera Charmus Karsch, 1879 and Reddyanus Vachon, 1972, stat n, Euscorpius 220, pp. 1-133 : 113-117

Hemispermatophores of the Buthids

We obtained new data on hemispermatophore morphologies of a variety of Old World buthids from Sri Lanka, that have not previously been described. Our findings may be significant in the context of the larger problem of defining monophyletic genera in the family and understanding relationships of major lineages of buthids. A preliminary buthid phylogeny was derived by Fet et. al. (2005) from the cladistic analysis of certain trichobothrial characters. They proposed a major family subdivision based on whether patella trichobothrium d 3 is positioned internal to the dorsomedian carina, which defined the ' Buthus ' group, or external to it, which is the character state for the remaining five 'non- Buthus ' groups. Hemispermatophores of ' Buthus ' group genera have been relatively well described, including for the genera Androctonus , Apistobuthus , Buthacus , Buthiscus , Buthus , Cicileus , Compsobuthus , Femtobuthus , Gint , Hottentotta , Leiurus , Lissothus , Mesobuthus , Microbuthus , Neobuthus , Odontobuthus , Orthochirus , Picobuthus and Vachoniolus ( Kovařík & Lowe, 2012; Kovařík, Lowe et. al., 2013; Levy & Amitai, 1980; Lowe, 2009, 2010a, 2010b, 2010c; Lowe et. al., 2014; Navidpour & Lowe, 2009; Vachon, 1952a, 1952b, 1958; Vachon & Stockmann, 1968). Their capsule region has a stereotypic 4-lobed configuration, in which the sperm hemi-duct is composed of 3 lobes (i.e. external lobe, carinated median lobe, and internal lobe), and a single basal lobe arises dorsally near the base of the median lobe carina. The flagellum is well separated from these lobes. So far, this '3+1' configuration has been found in all Buthus group members, suggesting that it is a synapomorphy for the group.

Hemispermatophores of non- Buthus group genera, including most of the Sri Lankan buthids (except Hottentotta tamulus ), are more heterogeneous. In the majority of genera, the base of the flagellum is broadly fused to a large carinated lobe, and there may be one or more additional distinct lobes on the internal side. A basal lobe is usually present, and its size and shape varies considerably. This configuration has been described in the genera Ananteris , Australobuthus , Butheoloides , Centruroides , Chaneke , Hemilychas , Isometroides , Isometrus , Parabuthus , Rhopalurus , Tityus and Zabius ( Botero-Trujillo & Florez-Daza, 2011; Francke & Stockwell, 198 7; Gysin & Le Coroller, 1968; Koch, 1977; Kovařík, Teruel, et al., 2015; Kovařík, Teruel & Lowe, 2016; Lamoral, 1979; Lenarducci et. al., 2005; Locket, 1990; Lourenço et. al., 2006; Maury, 1969, 1970, 1974; Ojanguren-Affilastro, 2005; Prendini et. al., 2009; Probst, 1972; Stockwell, 1989; Teruel & Armas, 2012). Here we document this lobe configuration also in the non- Buthus group genera, Buthoscorpio , Charmus , Lychas and Reddyanus stat. n.

Following Stockwell (1989), we have proposed that the carinated lobe of buthids is homologous to and derived from the carinated lobe of the chaerilid hemispermatophore ( Kovařík, Teruel & Lowe, 2016). In the ' Buthus ' group, this lobe sits between two other lobes with more external and more internal positions, and hence was termed the 'median lobe' ( Vachon, 1952). Taking the carina as a conserved landmark, we hypothesize that the median lobe is homologous to the carinated lobe in non- Buthus group genera that is fused to the base of the flagellum. We further hypothesize that the fused state is plesiomorphic because it approximates the chaerilid condition in which the carinated lobe is joined continuously to the distal lamina. Some exceptions to the fused configuration are seen in the genera Ananteris , Babycurus , Grosphus and Uroplectes ( Lamoral, 1979; Lowe, 2000; Kovařík, Lowe et. al., 2015, 2 0 16; Ojanguren-Affilastro, 2005;Vachon, 1950, 1969), in which the flagellum appears well separated from the carinated median lobe. However, in contrast to the Buthus group, a non-carinated external lobe is not interpolated between the flagellum and the median lobe. This arrangement could represent a precursor to the Buthus group configuration.

All Sri Lankan representatives of non- Buthus group genera so far examined ( Buthoscorpio , Charmus , Isometrus , Lychas and Reddyanus stat. n.) exhibit a basic 1+1 lobe configuration (fused median lobe + basal lobe), without additional internal lobes developed. This configuration is shared with a number of other non- Buthus group genera that have been reported, i.e. some species of Ananteris ( Botero-Trujillo & Florez-Daza, 2011; Ojanguren-Affilastro, 2005) and Tityus (Kovařík, Teruel, et. al., 2015; Ojanguren-Affilastro, 2005); and Australobuthus , Hemilychas , Isometroides and Isometrus ( Gysin & Le Coroller, 1968; Locket, 1990; Probst, 1972). This may be a plesiomorphic state from which additional internal or external lobes have arisen independently in different lineages. Even under a basic 1+1 configuration, we observe hemispermatophore differences that are diagnostic at the generic level, e.g. enabling us to differentiate Reddyanus from Isometrus by the size of the basal lobe, and the length and shape of the flagellum. Two novel, unique hemispermatophore morphologies that we found are: (i) a highly elongated capsule in Buthoscorpio , with a long narrow median lobe fused to the base of the flagellum, and a round, blunt basal lobe; and (ii) a short capsule in Charmus , with a truncated median lobe, and a bulging, bilobate basal lobe. These findings reveal an unexplored diversity in buthid hemispermatophores, that could provide new characters for analyzing phylogenetic relationships in this large and ancient family.

Cytogenetic Data on Sri Lankan Buthid Scorpions

Altogether we analyzed seven buthid species ( Figs. 547–554, Table 6) using standard cytogenetic methods (e.g. Kovařík et al., 2009; Šťáhlavský et al., 2014). The karyotype characteristics of all analyzed species correspond to the cytogenetic attributes typical for the family Buthidae . Chromosomes do not have visible centromere regions which is a typical feature for holocentric chromosomes. This type of organization is only known in the family Buthidae within scorpions (e.g. Mattos et al., 2013). This family is also characterized by low numbers of chromosomes. Half of the cytogenetically investigated species show diploid numbers from 14 to 24 (Schneider et al., 2016). In view of this fact, Charmus laneus (2n=9) ( Fig. 548) and Isometrus thwaitesi (2n=8) ( Fig. 549) represent exceptions with very low number of chromosomes. Only three known species from the genus Tityus C. L. Koch, 1836 have diploid numbers of chromosomes lower than 10 (see Schneider et al., 2016). This phenomenon has been documented in T. bahiensis (Perty, 1834) and was explained as an effect of intensive fusion of holocentric chromosomes ( Schneider et al., 2009). This type of chromosomal rearrangement may also explain the differences in chromosome size within Buthoscorpio sarasinorum ( Fig. 547), Lychas srilankensis ( Fig. 550) (both with one extra larger pair of chromosomes) and Isometrus thwaitesi ( Fig. 549) (with one extra shorter pair of chromosomes). Moreover, heterozygous chromosomal rearrangements may also explain odd diploid numbers of chromosomes in karyotypes of Isometrus thwaitesi ( Fig. 549), Reddyanus loebli ( Fig. 554) and one male of Reddyanus basilicus ( Fig. 552). In Reddyanus basilicus we found 2n= 16 in a male from locality 15CS. In this case the chromosomes gradually decrease in size ( Fig. 551, Table 6). However, the male of R. basilicus from locality 15CR has 2n=15, including one extra large chromosome ( Fig. 552, Table 6). The intraspecific variability has also been documented in another 8 species from different genera in the family Buthidae (see Schneider et al., 2016). Due to the intraspecific variability and high similarity of basic cytogenetic characteristics (such as number and size of chromosomes) it seems difficult to apply standard cytogenetic techniques to the taxonomy of buthid scorpions. In the future, application of more refined molecular cytogenetic techniques should lead to a better understanding of the organization of genome, which may be the key to detecting specific differences between closely related species.

Key to Scorpions of Sri Lanka

1. Pedipalp patella without ventral trichobothria. .......... Buthidae C. L. Koch, 1837 ..……………………......... 3

– Pedipalp patella with three ventral trichobothria. ...... 2

2. Adults 27–45 mm long. Pedipalp femur with 9 trichobothria, of which 4 are dorsal. ……....... Chaerilidae Pocock, 1893 , …... Chaerilus ceylonensis Pocock, 1894

– Adults 75–176 mm long. Pedipalp femur with 3 or 4 trichobothria, of which only one is dorsal ..................... Scorpionidae Latreille, 1802 ....…………………...... 15

3. Legs III and IV with well developed long tibial spurs ( Figs. 193–195, 198) .....……………………................ 4

– Legs III and IV without tibial spurs ( Figs. 196–197, 199) .......…………………............................................. 8

4. Telson with a distinct subaculear tooth ( Figs. 407– 408). ..........…........ Lychas srilankensis Lourenço, 1997

– Telson without subaculear tooth ( Fig. 421–427). ....... 5

5. Metasomal segments IV–V punctate without developed carinae ( Figs. 24–29). Dentate margin of pedipalp chela movable finger with distinct granules divided into 8–11 linear rows, apical rows of 3–6 granules, and 3 terminal granules ( Figs. 39–44). ...……......................... 6

– Metasomal segments IV–V not punctate with well developed carinae ( Figs. 120–121). Dentate margin of pedipalp chela movable finger with distinct granules divided into 13–15 linear rows and 5–6 terminal granules ( Fig. 46). ..... Hottentotta tamulus ( Fabricius, 1798)

6. Adults 25–52 mm long. Pedipalps, metasoma and telson glabrous ( Figs. 24–29). ..………….......................... ……...…….. Buthoscorpio sarasinorum ( Karsch, 1892)

– Adults 12–25 mm long. Pedipalps, metasoma and telson densely hirsute ( Figs. 71–73)..……………............ 7

7. Pedipalp patella yellowish with several black spots ( Figs. 118–119); metasomal segment V length/ width ratio is 1.28–1.43 in female ( Fig. 83); pedipalp chela length/ fixed finger length ratio is 1.69–1.79 in female ( Figs. 42–43)..……….... Charmus laneus Karsch, 1879

– Pedipalp patella black with several little yellow spots ( Figs. 111, 116); metasomal segment length/ width ratio is 1.80 in female ( Fig. 84); pedipalp chela length/ fixed finger length ratio is 1.45 in female ( Fig. 44). .............. ……………………………..... Charmus saradieli sp. n.

8. Chelal trichobothrium db located between dt and et. Fixed fingers of pedipalps with six rows of granules and six external and internal granules ( Figs. 252–253). Tarsomeres II of leg IV with two rows of dense setae ( Figs. 196–197). ........ Isometrus Ehrenberg, 1828 ….. 9

– Chelal trichobothrium db located between et and est ( Fig. 321). Fixed fingers of pedipalps with seven rows of granules and six external and seven internal granules ( Figs. 254–259). Tarsomeres II of leg IV with two rows of no more than 20 spiniform setae ( Figs. 199, 201– 208). ............ Reddyanus Vachon, 1972 stat. n. ….... 10

9. First (basal) middle lamella of pectine in both sexes rounded ( Fig. 558). Fingers and manus of pedipalp chela the same color, spotted ( Fig. 556). Posterior margin of sternite V strongly convex medially ( Fig. 560) …….. ….....………................……… I. thwaitesi Pocock, 1897

– First (basal) middle lamella of pectine in both sexes quadrangular ( Fig. 557). Manus of pedipalp yellow with several spots, fingers dark ( Fig. 555). Posterior margin of sternite V almost straight to very slightly convex medially ( Fig. 559). …..... I. maculatus ( De Geer, 1778)

10. Terminal tubercle of dorsal carina on second and third metasomal segment of male markedly enlarged ( Fig. 561). Pedipalp femur and patella spotted, patella mostly black ( Fig. 563), femur mostly yellow. Subaculear tooth spinoid ( Figs. 417–418). ..………...... …………………….. R. loebli ( Vachon, 1982) comb. n.

– Terminal tubercle of dorsal carina on metasomal segments of male are not enlarged ( Fig. 562). Pedipalps with brown spots, identical on femur and patella ( Fig. 564). Subaculear tooth wide and rounded ( Figs. 409– 416, 419–420). ….............……................................... 11

11. Glabrous zone on posterior part of fifth sternite present medially in male ( Figs. 569–570). ………….. 12

– Glabrous zone along posterior margin of fifth sternite absent or indicated ( R. besucheti ) ( Figs. 567–568). .... 13

12. Whole mesosoma dark, almost black ( Figs. 338– 339). Glabrous zone present in middle part of fifth sternite only ( Fig. 569). ....……..... R. jayarathnei sp. n.

– Mesosoma lighter, yellowish brown ( Figs. 382–383). Glabrous zone stretches almost over whole posterior margin of fifth sternite ( Fig. 570).... R. ranawanai sp. n.

13. Manus of pedipalp chela wide in male. Ratio pedipalp chela length/width 2.94–3.28 ( Fig. 566, Table 5). Chela wider in male than in female. .......…........... 14

– Manus of pedipalp chela narrow in male. Ratio pedipalp chela length/width 3.41–3.79 ( Fig. 565, Table 5). Chela the same width in both sexes. .................... ………......……… R. basilicus ( Karsch, 1879) comb. n.

14. Ratio metasomal segment II length/width 1.56–1.79 in male ( Fig. 211, Table 5). ............................…........... ………...………. R. besucheti ( Vachon, 1982) comb. n.

– Ratio metasomal segment II length/width 1.85–1.97 in male ( Fig. 213, Table 5). ……….... R. ceylonensis sp. n.

15. Adults 128–176 mm long. Pectinal teeth number 16– 20. Fifth segment of metasoma longer than pedipalp femur, fourth segment of metasoma about as long as pedipalp femur ..…........ H. swammerdami Simon, 1872

– Adults 75–130 mm long. Pectinal teeth number 10–16. Fifth segment of metasoma about as long as pedipalp femur, fourth segment of metasoma shorter than pedipalp femur. .....…………………………………….... 16

16. Pedipalp chela with carinae on dorsoexternal surface ( Fig. 572)..………......... H. gravimanus ( Pocock, 1894)

– Pedipalp chela without carinae on dorsoexternal surface ( Fig. 571). .......…………..................................... 17

17. Dorsal and dorsolateral carinae of metasomal segments smooth ( Fig. 573). ….. H. indus ( De Geer, 1778)

– Dorsal and dorsolateral carinae of metasomal segments granulated ( Fig. 574). ..….... H. serratus ( Pocock, 1900)