Malagasy Polka Dot Moths (Noctuoidea: Erebidae: Arctiinae: Syntomini) of Ambohitantely—endemism in the most important relict of Central Plateau rainforest in Madagascar

Malagasy Syntomini (Polka Dot Moths) are one of the largest endemic lineages of Lepidoptera on the island, belonging to the Tiger Moth subfamily (Arctiinae). This diverse radiation comprises nearly 100 valid described species that share a single ancestor. Despite a monograph in 1964 by Paul Griveaud, systematics of the group greatly needs modern revision, and their distribution on the island is still poorly known. This contribution concerns the diversity of Syntomini of the Réserve Spéciale d’Ambohitantely, which protects the largest remaining, but already highly fragmented, vestige of Central Plateau rainforest in Madagascar. Here we provide an annotated checklist of the eight species occurring in the Reserve. Two species are recorded from the forest for the first time, while five endemics are until now known only from Ambohitantely. We also describe for the first time the female of Thyrosticta vestigii Griveaud, 1964 and of Maculonaclia tampoketsya Griveaud, 1969, as well as a yellow morphotype of Thyrosticta dilata Griveaud, 1964, and we redescribe and illustrate the genitalia of the remaining species. The significance of such colour pattern variation in aposematic moths and the role of this Reserve as a local centre of diversity of Malagasy Syntomini together with its importance in the protection of the biodiversity of Madagascar are discussed.

158 Canon 70D camera before the further examination. The images were adjusted with Adobe 159 Photoshop. Specimens are deposited in the collection of ISEA PAS, Kraków, Poland, accession 160 numbers of the specimens are provided in Supplemental Table S1. 161 Genitalia were dissected from one specimen of each sex of collected species, except for 162 males of Tsarafidynia perpusilla, where two slides of the same sex were prepared. Abdomens 163 were macerated in 10% KOH in water bath, then obtained genitalia were stained with chlorazol 164 black, embedded in Euparal (Essex, U.K.) and mounted on slides. Photographs of the genitalia 165 were taken with the use of a stereoscope microscope Leica S9i system. Images were adjusted 166 with the Adobe Photoshop Programme. 167 The general morphological terminology follows Miller (1991), and for genitalia we refer 168 to Koda (1987). Measurement of forewing length (in mm) was taken with the use of a digital 169 caliper. 170 We summarise our results with updated data published on distribution and ecology of 171 Syntomini species occurring in the Réserve Spéciale d'Ambohitantely. Official names of 172 protected areas mentioned in the text follow Goodman, Raherilalao & Wohlhauser (2018). The 173 most important collections of Malagasy Syntomini, including type specimens of majority of the 174 species, are deposited in three institutions: MNHN in Paris, NHMUK in London and PBZT in 175 Antananarivo. However, we may have missed some information, as in the latter collection, red 176 paratype labels are pinned under the main labels, thus in available photographs were visible 177 partially, if at all. Moreover, some species have more specimens labelled as paratype than 178 designated in , even taking into consideration only London and Paris collections, 179 where the photographed labels are clearly visible. These last collections were inaccessible for 180 examination at the time of writing (due to . Thus, to make some morphological and 181 taxonomical remarks and to confirm localisation of type specimens of species that are dealt with 182 in the paper, we relied on photographs of specimens and genital slides taken by ŁP in MNHN in 183 189 For molecular investigation, two legs from each specimen were sampled. Isolation of genomic 190 DNA was done with the NucleoSpin Tissue kit (Machery-Nagel, Germany), following the 191 manufacturer's protocol. Sequences of the first part of the mitochondrial gene cytochrome c 192 oxidase subunit I (COI) were obtained with the use of HCO/LCO primers pair hybridised with 193 the universal primer pair T7/T3, described by Wahlberg & Wheat (2008). PCR was done with 194 the use of hot-start ready PCR mix (StartWarm HS-PCR Mix, A&A Biotechnology, Poland), 195 protocol followed manufacturer's instructions. Obtained sequences were compared with 196 chromatograms, aligned manually with a template sequence in BioEdit software (Hall, 1999). 197 Ambiguous sites were coded in accordance to the IUPAC nucleotide code.  . Holotype and three 249 paratypes are deposited in MNHN, one paratype is in NHMUK. Further three specimens are in 250 PBZT, and their collecting data labels are identical to these of specimen in Paris and London, 251 thus they probably are remaining paratypes. Among specimens labelled as paratype of 252 Maculonaclia altitudina in Paris there is one additional specimen, undoubtedly belonging to the 253 species Maculonaclia brevipenis, that is similar in general appearance, but distinctly differs in 254 details of forewing pattern. As discussed below, the type series of Maculonaclia brevipenis in 255 Paris contains many more specimens labelled as paratypes than stated by , and 256 all the specimens of both species were collected in the same place, at the same time and by the 257 same collector.

258
All the known specimens of Maculonaclia altitudina were collected in May, in the cool 259 dry period. Papillae anales subtriangular with rounded protrusion at base of dorsal margin, covered with 275 short erected setae, much denser on the protrusion; dorsal and ventral pheromone glands present 276 in form of very narrow, elongate, not anastomosing membranous tubes; apophyses posteriores 277 almost as long as papillae anales, straight and narrow, needle-like; apophyses anteriores of 278 similar shape and size as apophyses posteriores; ostium bursae rounded; antrum well developed, 279 sclerotised, cylindrical, slightly longer than wide; ductus bursae membranous, slightly widening 280 towards corpus bursae, terminal portion with sublateral diverticulum directed distally, from 281 which narrow, membranous ductus seminalis originates; 282 corpus bursae forming a membranous, oval pouch bearing indistinct, irregular zones of minute, 283 diffuse scrobinations; central portion with a pair of signa in form of short, parallel ridges 284 consisted of tiny subtriangular sclerotised plates, leaning on each other; along a longitudinal axis 285 designated by the signa, scrobinations are slightly strongly articulated. 286 Remarks 287 Male and female genitalia were described and illustrated by Griveaud (1964: Figs 87-90). 288 Corpus bursae is depicted to possess scrobinations only in the rhomboidal areas surrounding 289 each of two signa. In the genital slide of allotype (MNHN) these areas are indeed more 290 prominent than in the slide prepared from our specimen (Fig. 4A), where minute scrobinations 291 are diffuse over whole corpus bursae, and only slightly larger around signa. It could be a matter 292 of intraspecific variation, but also an effect of different staining technique, as slides of Griveaud 293 are prepared with eosin, whereas our ones with chlorazol black. This issue needs further 294 examination in the future on larger series of specimens.

295
Type series of Maculonaclia ankasoka designated by  is given to 296 comprise 10 specimens: male holotype, and nine paratypes (one male and eight females of which 297 one labelled as allotype). The holotype and the male paratype were collected by P. Griveaud in 298 November 1956 in Ankasoka at an elevation of 1000 m (however original label of the holotype 299 says "1130 m"), the allotype and the remaining female paratypes in February 1961 at Périnet, 300 elevation 900 m . The holotype, allotype and three other paratypes are deposited 301 in MNHN. In Paris are also other seven specimens: four collected by P. Griveaud and R. Vieu in 302 1956 and three collected in 1959, 1963 and 1964 by P. Viette (detailed collecting data illegible in 303 the photographs). A further about 30 specimens determined as Maculonaclia ankasoka are in the 304 PBZT collection, collected mostly by P. Griveaud. Three of them are most probably remaining 305 paratypes, as their labels agree with data given by   (Fig. 5A) 315 Tegumen narrow, moderately sclerotised, almost completely fused with vinculum; uncus 316 elongate, dorso-ventrally flattened, slightly concaved in ventral surface; of the same width up to 317 sharply narrowed, ventrally incurved hook-like tip; dorsally covered with erected setae, longer in 318 basal portion; vinculum narrow produced with a prominent saccus of triangular shape; juxta well 319 developed, divided into transverse ventral plate and a pair of lateral, rectangular plates; valva 320 approximately the length of uncus with terminal half of triangular shape; costa evenly convex, 321 widely folded towards inner zone; tiny, tooth-like protrusion in the 1/3 of folded costal margin; 322 saccular margin shallowly sinusoidal; margins and some regions of internal and external surface 323 with short erected setae; aedeagus weakly sclerotised, short, tubular, slightly narrowing towards 324 apex; vesica membranous, bag-like, with four small sclerotised plates of irregular shape in 325 latero-distal portion. 326 Remarks 327 Male genitalia were described and illustrated by  : Figs 95-97). Figures show 328 valva with sharply terminated apex, narrow elongate saccus, and vesica was uneverted. In fact 329 the valva is dully terminated and saccus is triangular, but not elongate, vesica as described above.

330
Until now the species has been known from the type series, according to  331 consisting of the male holotype and two paratypes, collected by A. Robinson in May 1961 at an 332 elevation of 1550 m. The female remains unknown. However, in MNHN, except the holotype, 333 are deposited 10 specimens marked as paratypes, labelled with identical collecting data as given 334 above. Further two paratypes are deposited in NHMUK. Another 13 specimens with identical 335 labels are in PBZT collection, at least one of which is also labelled as a paratype, because a 336 fragment of a red label is visible from under the collecting data label.

337
All the known specimens were collected in December, March and May, during the warm 338 rainy period and at the beginning of the cool dry period.  (Fig. 5B) 347 Tegumen completely fused with vinculum, very narrow, moderately sclerotised, with a pair of 348 prominent, sharp, claw-like protrusions directed ventrally, close to the uncus base; uncus large, 349 elongated, bent ventrally, laterally flattened, with tiny spike-like protrusion at the tip; basal half 350 with numerous long setae; saccus short, terminated with tiny, narrow protrusion; valva elongate, 351 reaching almost to uncus tip, narrowed terminally into sclerotised, hook-like process slightly 352 curved ventrally; costal margin widely sclerotised, concaved submedially, with some undulation 353 in its basal portion; concavity marked with a narrow, membranous, joint-like articulation; 354 sacculus sclerotised, reaching till 2/3 of valva length, with short erected setae, extending beyond; 355 a short spike-like protrusion at dorsoterminal margin beyond sacculus; central inner portion of 356 valva membranous; aedeagus tubular, widened subbasally, slightly bent dorsally in distal 357 portion; vesica in form of membranous tube evenly widened in proximal 2/3 of its length, 358 bearing a dense bunch of elongate, needle-like cornuti in terminal portion.
Remark: The short spike-like protrusion at dorsoterminal margin beyond sacculus visible 360 only on right valva. Left valva with indistinct convexity. 361 Description of female (Fig. 3C) 362 Head. Proboscis well developed, brown, apex and base pale brown; frons pale yellow, with 363 longitudinal ochraceous stripe from clypeal portion towards second third; vertex ochraceous with 364 admixture of pale yellow scales, lateral margins yellow, ochraceous stripe between scapi; palpi 365 three-segmented, porrect, yellow, ventrally with elongate scales, dorsally with admixture of 366 ochraceous scales, terminal palpomere dorsally entirely ochraceous; antennae filiform, 367 ochraceous with admixture of creamy scales, except terminal, dark ochraceous quarter. 368 Thorax. Patagia of piliform scales, submedially ochraceous with tiny yellow spot in central 369 portion, laterally pale yellow; tegulae pale yellow with elongate scales almost piliform in distal 370 portion, terminally with admixture of ochraceous; subventral zone ochraceous; mesothorax 371 ochraceous, medially with longitudinal narrow yellow stripe and yellow spot in distalomedian 372 portion; metathorax ochraceous; ventral portion of pleurites ochraceous, with yellow blotches at 373 base of coxa; foreleg: pale yellow, epiphysis absent; midleg: pale yellow, tibia with one pair of 374 terminal spurs of similar length; hindleg: coxa and femur pale yellow; remaining parts of the 375 hindleg unavailable. 376 Abdomen. Ochraceous, distal margin of each segment with yellow stripe. 377 Forewing. Length of costa 11mm (n=1); upperside background ochraceous, with short, yellow, 378 narrow streak along proximal portion of dorsum and additional 5 pale yellow to creamy blotches 379 of subrectangular shape and similar size: 1 at basal 2 at medial and 2 at distal portion of wing; 380 basal one elongate, from costal margin to the half of the wing width, with a prominent narrow 381 projection towards wing base on R vein; first medial one of rectangular shape, form costal 382 margin to hind margin of DC; second medial one of irregular shape, from cubital vein, widening 383 towards termination before inner margin; first distal one elongate, from costal margin to M3, 384 constricted in medial portion along M1; second distal one below CuA1, of irregular shape, 385 separated from outer margin by narrow ochraceous stripe; underside with the same pattern, with 386 addition of zone of scattered pale yellow scales between the basal blotch and 1A+2A; cilia 387 ochraceous. 388 Hindwing. Elongate, reaching about half of forewing; basal portion yellow, reaching to the 389 basalodistal angle and to 3/4 of the length of costal margin, with large, 8-shaped elongate 390 ochraceous blotch, originating from the wing base and including most of DC, but not reaching to 391 its outer margin nor the costal margin of the wing; outer area ochraceous; underside pattern the 392 same but lateral portion of brown blotch reaches the costal margin; piliform scales along wing 393 margins, longer on basalodistal margin; frenulum present. 394 Female genitalia (Fig. 4B) 395 Papillae anales semicircular, covered with short, dense, erected setae; dorsal pheromone glands 396 present in form of narrow, rather stright, not anastomosing tubes, of about three lengths of 397 apophyses posteriores; apophyses posteriores strongly sclerotised, straight and narrow, needle-398 like; apophyses anteriores in form of subtriangular, short lobes, half of the length of apophyses 399 posteriores; 7th and 8th segments heavily sclerotised; 7th sternite wide and narrow with The black morphotype distinctly differs from the yellow one in the characters listed 521 below (Fig. 3E-F): head (including palpi and antennae) and thorax (including patagia) are 522 entirely black, tegulae yellow with piliform scales. Legs are fully dark ochraceous, including 523 spurs. Forewing has the same shape and pattern as in yellow form, but elongate streak along 524 costal margin is always absent. Abdomen is entirely black dorsally and ventrally, first tergite 525 possess elongated black scales.

526
As already mentioned, no intermediate form of Thyrosticta dilata has been detected, 527 however both morphotypes exhibit internal variation in the colouration described below.

528
In the yellow morphotype (n=22) ochraceous scales on frons, close to eyes margin, are 529 absent in some specimens. The ochraceous stripe on vertex varies from a very narrow band to a 530 globular blotch, reaching or not to the black stripe between antennae. Elongate streak on costal 531 margin of fore wing reaches half of the wing and fuses with the second U-shape blotch or 532 terminates before. In some specimens also comma-shape projection of apical blotch reaches 533 close to or fuses with DC blotch, up to fuse of these three blotches, creating a yellow stripe along 534 costal margin fused with them. When the wings pattern is strongly developed, U-shaped (the 535 largest) and round (the second) blotches nearly touch each other, but never fuse.

536
In the black morphotype (n=23) some specimens have general body colouration in dark 537 ochraceous rather than blackish. In some specimens with strongly developed wings pattern U-538 shaped (the largest) and round (second) blotches nearly touch each other, up to fuse.

539
The species has until now been known only from the type series designated by Griveaud 540 (1964), declared to consist of the male holotype and four paratypes. The female remains 541 unknown. The holotype was collected by P. Griveaud on 27.xii.1956 at an elevation of 1600 m. 542 Paratypes are said to have been collected in May 1961 and to have the same provenance and 543 collector as the holotype , but according to their labels, all the five specimens 544 from May 1961 were collected by A. Robinson at an elevation of 1550 m, not by P. Griveaud at 545 1600 m. 546 The holotype and one paratype are deposited in MNHN, another paratype is in NHMUK. A 547 further three specimens with labels identical as these of the paratypes in Paris and London are in 548 PBZT, thus most probably among them are remaining two paratypes. In PBZT there are also 549 additional four specimens, collected in April 1967 by P. Griveaud (two specimens), in October 550 1974 by A. Peyrieras (one specimen) and in 1970s (one specimen, exact year and name of 551 collector illegible in the photograph). All of them were collected in the area of Tampoketsa 552 d'Ankazobe as well, however the specimen from 1974 remains uncertain because of illegible 553 locality on the label, except "central Madagascar".

554
For the reason given below, we assume that Griveaud was aware of the intraspecific 555 variation when describing the species, but for some reason omitted it. The original description 556 and colour illustration  pl. I, Fig. 60) refer to the black morphotype. However, 557 the holotype deposited in MNHN represents the yellow morphotype, while the paratype in the 558 same collection belongs to the black one. Genitalia were described and illustrated in Griveaud 559 (1964: Figs 224-226), but with an uneverted vesica on the aedeagus, which is described above.
As indicated in the Materials section, almost all of the fresh specimens of Thyrosticta 561 dilata were collected at light traps with both UV or non-UV white light sources, which allowed 562 us to obtain series of well-preserved specimens. According to our observations, this is rather 563 exceptional among Malagasy Syntomini, although DCL has observed it for some members of 564 genera Thyrosticta and Tritonaclia at other sites. As a general rule, syntomines are attracted to 565 light rather rarely and usually just in small numbers, which makes day netting the most efficient 566 collecting method for the vast majority of taxa.  (Fig. 3D) 575 Head. Entirely blackish ochraceous, including palpi and antennae; palpi projected downward; 576 proboscis well developed; antennae filiform, flagellum with numerous short, erected setae. 577 Thorax. Concolorous with head both dorsally and ventrally, including patagia and filiform 578 tegulae; metascutellum with partially filiform scales; legs entirely blackish ochraceous, with 579 exception of paler epiphysis on foreleg; mid and foreleg tibia with one pair of terminal spurs. 580 Abdomen. Entirely blackish ochraceous dorsally and ventrally, with admixture of piliform 581 scales. 582 Forewing. Length of costa 7 mm (n=1); upperside blackish ochraceous, with two partially fused 583 yellow blotches; first one prominent, reaching from the wing base up to half of the wing length 584 terminating at DC outer margin; costal portion along R stem with indistinct, shallow, concavity 585 in its half-length; opposite margin in proximal part along narrow ochraceous streak of inner 586 margin of wing, in distal part directed to a right-angle-shape blotch termination; second blotch in 587 postdiscal zone, of dumbbell-shape, fusing narrowly in inner posterior angle with the tip of first 588 blotch; cilia and scales along inner margin elongate concolorous with background; underside 589 pattern the same. 590 Hindwing. Oval, elongated, reaching beyond the half of fore wing; basal part including DC with 591 yellow oval zone, reaching to the basalodistal angle and beyond the half of the costal margin; 592 outer zone brown, with narrow brown margin along costa; underside pattern the same, with 593 addition of short brown protrusion from brown costal margin towards central part of yellow 594 zone; elongated scales on outer and hind margins, with dominance of piliform scales close to 595 wing base; frenulum present. 596 Female genitalia (Fig. 4C) 597 Papillae anales subtriangular, covered with erected setae, much denser and longer in ventral 598 portion; dorsal pheromone glands well developed, in form of four very narrow, elongate, twisted, 599 rarely anastomosing membranous tubes; two sublateral much longer than two submedial;