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DNA barcodes from over-a-century-old type specimens shed light on the taxonomy of a group of rare butterflies (Lepidoptera: Nymphalidae: Calinaginae) [1]

['Valentina Todisco', 'Department Of Environment', 'Biodiversity', 'Paris Lodron University Of Salzburg', 'Salzburg', 'Dipendra Nath Basu', 'National Centre For Biological Sciences', 'Tata Institute Of Fundamental Research', 'Bengaluru', 'Sean W. J. Prosser']

Date: 2024-08

Although many studies show the importance of combining different markers to obtain robust species-level identification [34], COI barcode sequences have proven to be sufficient in differentiating species in a wide range of organisms [35]. In this study our morphological analyses in conjunction with COI and RPS5 sequence data shed light on the long-standing problem of the systematics in genus Calinaga.

Despite evidently being a geologically old genus [24, 36, 37], Calinaga does not demonstrate nearly the same degree of species divergence and differentiation that is prevalent in other nymphalid groups of similar age. This pattern is typical of “living fossils”, ancient lineages with one or few living representatives, such as the Mexican Baronia brevicornis, the lone species in a monotypic subfamily that is sister to all other swallowtails [38]. Similarly, the limited morphological differentiation in wing pattern, combined with individual variation and clinal gradients, has been the main source of historical confusion about the systematics of Calinaga.

Taxonomic consideration

Ever since its 19th century description by Frederic Moore, there has been much taxonomic uncertainty regarding not only the higher classification of Calinaga within the butterflies, but also, as the diversity of named phenotypes included in the genus increased, the relative, species-level status of its component taxa. Ehrlich [7, 18] effectively anticipated the solution to the higher classification problem (the Calinaginae are now convincingly regarded as the sister group of the remainder of the satyroid clade, within Nymphalidae sensu lato [36, 37]), but Ehrlich was non-committal on the species diversity issue: He simply treated Calinaga as a single, polytypic species. Ever since, the number of Calinaga species to be recognised has remained very uncertain.

In his recent monographic account, Tshikolovets [25] divided Calinaga into five superspecies: buddha, brahma, formosana, davidis and lhatso–and among these, a total of 16 named species and semispecies (some with several subspecies).

At superspecies level, Tshikolovets’s classification is largely congruent with our results, but with some exceptions. We found the following groups:

I-a. Calinaga lhatso. Under this group, Tshikolovets lists lhatso, senseiensis, pacifica, dubernardi, funebris and funeralis as valid “semi-species”. Our results show that taxa lhatso, pacifica, and funebris undoubtedly belong to this clade. The oldest name in this clade is lhatso Oberthür, 1893 and therefore we recognize this clade as Calinaga lhatso with subspecies lhatso and funebris stat. nov. Our single specimen of pacifica appeared as sister to the remaining samples in this clade and may perhaps later prove to be a “good” species; however for now we consider it part of the variation in this clade. The little-known taxon dubernardi Oberthür, 1920 (TL: “Tsekou” [Cigu, Deqin, Yunnan]), provisionally included in this group, has no surviving type material, but according to the original description it shows intermediate characteristics between lhatso and brahma; its type locality is the same as lhatso and it is possibly a form of it or a hybrid between lhatso and brahma [25]. In our phylogenetic analysis, the taxa senseiensis (one specimen) and funeralis (holotype and one paratype) did not appear to belong to this group but to brahma; however for now we maintain their position in the lhatso group as incertae sedis until further evidence to support their position becomes available. We could not sample the taxon yaonica, but morphologically it appears to belong to this group and be closely related to funeralis.

I-b. Calinaga buddha. Our results largely support the taxonomy proposed by Tshikolovets [25] for this group, who recognized three taxa within it: buddha, avalokita and gautama. The C. buddha avalokita specimens sequenced in this work carry location labels of Silhet (now in Bangladesh) and Thailand. Both these are doubtful since Silhet does not have the elevation and habitat suitable for Calinaga, and the supposed Thailand source lacks details. Thus, the true location of avalokita is uncertain. The specimens were collected perhaps in Meghalaya, Patkai Hills or nearby ranges, and brought to the British outpost in Silhet. Even though in our results this group is not monophyletic, the samples always cluster together in a closed group. Since no geographic distinction could be inferred from our phylogeny supporting separation of these taxa at species level, we recognize Calinaga buddha with three subspecies: buddha, avalokita and gautama stat. rev. The placement of gautama as a subspecies of C. buddha is tentative and requires further taxonomic study.

I-c. Calinaga brahma. Tshikolovets [25] recognized sudassana, nicevillei, bedoci, and distans under brahma as well as two new subspecies (nujianga and lancangjianga). Our results confirm that these taxa do indeed fall within the brahma clade. Even though DNA barcodes do not discriminate between any of these taxa, they all appear to be allopatric in distribution and potentially represent valid subspecies (Fig 2). We therefore recognize them as subspecies of brahma (but see above for taxa senseiensis and funeralis).

Diagnosis. This new subspecies shares at least three morphological characters with typical Calinaga aborica that link the two taxa together (Clade II), while separating them from all other known Calinaga: red hairs on the thoracic dorsum restricted to the tegulae, hindwing discocellular veins m1–m2 and m2–m3 form a slight but distinct obtuse angle (ca 150–160°), and postdiscal and submarginal pale spots in hindwing cells M3, CuA1 and CuA2 uniquely arranged (Fig 6a and 6b(i); see S3 File for more information). Subspecies naima differs from the nominotypical, western subspecies in generally having a much ‘brighter’ wing-pattern–although, as with subsp. aborica, there is considerable individual variation in all pale markings. Two colour pattern features are considered diagnostic: In subspecies naima, on the underside, the dark ground colour is relieved by numerous yellow scales, these being dense in many areas, including the bases of hindwing cells CuA 1 and CuA 2 . In contrast, these yellow scales in subsp. aborica are far less dense, including those at the bases of hindwing cells CuA 1 and CuA 2 , which areas are thus far darker.

and CuA . In contrast, these yellow scales in subsp. aborica are far less dense, including those at the bases of hindwing cells CuA and CuA , which areas are thus far darker. The subtriangular, submarginal pale spot in cell M 1 on the hindwing upperside varies from being slightly wider than, to about the same width as the postdiscal streak in the same cell, such that if virtual anterior and posterior tangential lines are drawn to touch these two marks, the lines do not converge before the base of the wing, being more or less parallel (Fig 6b(ii)). In subsp. aborica such virtual lines converge before the base of the wing (Fig 6a(ii)). PPT PowerPoint slide

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TIFF original image Download: Fig 6. a) Calinaga aborica aborica (Lectotype). (i) Divergent centres of submarginal and postdiscal spots in HW cells M 3 , CuA 1 and CuA 2 . (ii) Anterior and posterior tangential lines touching the main postdiscal streak and submarginal spot in HW cell M 1 converge well before the base of the wing. b) Calinaga aborica naima (paratype 010243161). (i) Divergent centres of submarginal and postdiscal spots in HW cells M 3 , CuA 1 and CuA 2 . (ii) Anterior and posterior tangential lines touching the main postdiscal streak and submarginal spot in HW cell M 1 , even if converging, do not converge before the base of the wing. Note also how, in subsp. naima, the yellow scales in HW cells 1A and 2A are more dense, and extend almost to the wing margin. https://doi.org/10.1371/journal.pone.0305825.g006 Typically in subsp. naima (Fig 5), in comparison to subsp. aborica, the pale postdiscal streaks in forewing upperside cells R 5 , M 1 and M 2 are slightly better developed (streak in cell R 5 ca 3–4 mm); the pale streaks in hindwing upperside cell R 1 are generally far more prominent; the distal area of hindwing upperside cells 2A and 3A are often pale yellow almost to the margin (Fig 6b, cf. Fig 6a); and on the hindwing underside, the distal triangular spot in cell R 1 is always present, usually larger and less dyslegnic (i.e., having its margins more sharply defined), about 2.5–5.5 mm in length. Forewing length (male): mean 40.96 mm [n = 17; observed range 37.6–43.1 mm, SD 1.441]; female unknown. Material directly examined: holotype and 12 paratypes in NHMUK, London Photographs examined: 3 paratypes in the collection of the late Prasobsuk Sukkit (courtesy of Adam Cotton, Chiang Mai) and 1 paratype, ex Sukkit collection, in the collection of Howard Grisham, Alabama. Another specimen ex Sukkit collection, a specimen from ‘Wanzewong-Ngawar’ figured by Shizuya et al. [40]; and an online image of a male from ‘Chudu Razi Hills’ [see S3 File], are not included as paratypes.

Holotype ♂. Myanmar / Upper Burma: Seinghku Valley, 6500′, 28.5 N 97.35 E., 27.v.1926, F. Kingdon Ward. / Brit. Mus. 1926–400 / BMNH(E)#985060 / B.M. Type No. Rh. 17202 / Calinaga aborica naima Vane-Wright Holotype ♂ det. R.I. Vane-Wright 1971 / NHMUK. [See S3 File on Kingdon Ward’s localities.]

Paratypes. Myanmar–1 ♂ / Upper Burma: Seinghku R.[iver], 28.3 N, 97.31 E, 5000′, 17.v.1926, F. Kingdon Ward [see Kingdon Ward, 1930] / NHMUK. 3♂♂ / N. Burma: Adung Valley, 6000′, 14.v.1931, Lord Cranbrook [one specimen barcoded] / NHMUK. 2♂♂ / Mairudam, N Kachin State, Burma Prasobsuk Sukkit leg. 5 May 1998 / 5.v.1998 N.E. Putao. Kachin State Myanma [sic] Sukkit & Nishimura Coll. / 1♂ / Mairudam, N Kachin State, Burma Prasobsuk Sukkit leg. 14 May 1998 / 14.v.1998 N.E. Putao. Kachin State Myanma [sic] / Sukkit & Nishimura Coll. 1♂ / Mairudam, 12.v.1998 N.E. Putao. Kachin State Myanma [sic] leg. Sukkit & Nishimura / Howard Grisham Coll. India–3♂♂ Assam, Mishmi Hills, 1928, Percy Sladen Expn. / 2♂♂, Assam, Mishmi Hills, 2000′, 3.iii.1928, Percy Sladen Expn. / 28°0′ N. 96°0′ E / [one specimen barcoded] /, 1♂, Assam, Mishmi Hills, 4500 ft, 11.v.1928, Percy Sladen Expn. / Delei Valley [see Kingdon Ward, 1930], 28°21′ N, 96°37′ E, 4500ʹ, 11/5/28. / 2♂♂, Assam, Mishmi Hills, 10000 ft, 13.vi.1928, Percy Sladen Expn. / 28°21′ N, 96°37′ E /. [all NHMUK] Distribution: India–Arunachal Pradesh: Mishmi Hills. Myanmar–N Kachin State: Seinghku Wang Valley, Adung Valley, Mairudam (NE Putao), Wanzewong-Ngawar (north of Putao), Chudu Razi Hills (ca 40 km east of Kawnglangphu). [For details, including estimated coordinates, see S3 File.] Flight period and altitudinal range: records are for March–June; altitude 600–3000 m (mid-montane).

Etymology and nomenclature. Named after the ballad ‘Naima’, originally composed and performed by saxophonist John Coltrane for his 1960 album Giant Steps. The name was chosen (and the NHMUK specimens so-labelled) in 1971, but it has not been formally established until now. The name naima has, however, appeared in the literature at least three times as a nomen nudum or manuscript name [11, 25, 39]. While it is sometimes best to avoid subsequent establishment of a nomen nudum, in this case little purpose would be served by introducing a novel alternative; Calinaga aborica naima is here established with Vane-Wright (in Todisco et al. [24]) as author, and should be dated from the publication date of this paper (ICZN [41]: glossary).

III. Calinaga formosana. This isolated taxon from Taiwan, which was not included in the analysis of Todisco et al.’s [24], appears as a well-supported, distinct clade. We confirm its undisputed status as a valid species (see also S4 File), with no apparent sub-population structure.

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[1] Url: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0305825

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