Revision of Pazala Moore, 1888: The Graphium (Pazala) alebion and G. (P.) tamerlanus Groups, with Notes on Taxonomic and Distribution Confusions (Lepidoptera: Papilionidae)

Three Graphium species belonging to two species groups of the subgenus Pazala , the alebion and tamerlanus groups, were examined in molecular and morphological studies, and their female genitalia are reported for the first time. Their relationship with other species groups within the subgenus is assessed and their divergence times are estimated. We find that G. ( P. ) alebion is the first lineage to diverge within Pazala in the early Miocene (20 Ma) and that G. ( P. ) tamerlanus and G. ( P. ) parus are sister species and diverged from each other in the late Miocene (7 Ma). A revision of the four recognised taxa belonging to three species is presented, and historical misidentification of these taxa and their geographic ranges are explained.


Introduction
The Graphium (Pazala) alebion and G. (P.) tamerlanus groups are two species groups of subgenus Pazala Moore, 1888 as defined by Hu et al. (2018). They currently consist of only three species, namely G. (P.) alebion (Gray, [1853]) in the alebion group; and G. (P.) parus (Nicéville, 1900) and G. (P.) tamerlanus (Oberthür, 1876) in the tamerlanus group. Both groups are in relatively 'primitive' phylogenetic positions compared to the mandarinus, eurous, and mullah groups . The shared morphological characters include a single, non-bifurcated hindwing discal band ("8"-shaped in the mandarinus group, double paralleled in the eurous group, and posteriorly bifurcated in the mullah group), as well as simple, smaller, triangular-or pear-shaped dorsal terminal harpe in the male valvae (Racheli & Cotton 2009). However, the female genitalia of these three species, which can provide new insights in taxonomy of Pazala, have not been systematically described and compared to date.
During the taxonomical revision of subgenus Pazala, we carefully re-examined the types of all these three species as well as other available materials in the Natural History Museum (BMNH, London, UK) and the Höne collection of the Zoologisches Forschungsinstitute und Museum Alexander Koenig (ZFMK, Bonn, Germany). Freshly collected specimens were also carefully examined, dissected, and used for molecular phylogenetic analyses. Although our analyses did not discover any cryptic species or new subspecies like the preceding studies Hu et al. 2019), some note-worthy historical, taxonomic, and distributional confusion in museum collections and literature were discovered. 78 · Zootaxa 4759 (1) © 2020 Magnolia Press The aims of the present study are the reconstruction of phylogenetic relationships of known species and subspecies of the two groups, estimation of molecular divergence times of all recognised taxa of the two groups, and comparison of male and female genitalia. A distribution map and notes addressing the taxonomic and distributional confusions are discussed after the systematic study.

Taxon sampling
A large number of specimens of all taxa in the alebion and tamerlanus groups were examined and sampled from the authors' private collections, other private collections and specimen depositories of academic institutions, with permission. A list of all the examined materials of taxa in the alebion and tamerlanus groups, including type specimens, together with the names and abbreviations of depositories, is given in Appendix 1 at the end of this work. Subspecific designations for Pazala species followed Racheli & Cotton (2009).

Morphological comparison
Freshly obtained specimens were spread for examination, with the scent scales on their hindwings exposed.
Spread specimens were photographed using a digital camera with medium grey background. Photos were adjusted using Adobe Photoshop CS (Adobe, USA). For comparison between taxa, the lengths of forewing were measured to 0.5 mm precision. Average lengths of forewing were calculated, and the standard deviations were also calculated when n ≥ 3.
To observe the male and female genitalia, the abdomen was removed from the specimen and placed into a 1.5 mL microcentrifuge tube, and 1 mL water was added to the abdomen to rehydrate the tissue at 50 °C for 30 min, then 1 mL 10% sodium hydroxide solution was used to digest soft tissue at 70 °C for 1 h. The treated abdomen was neutralised with 2% acetic acid and then dissected in a water-filled Petri dish under the stereoscope to remove residual tissues, scales, and hair. The genitalia were then transferred to 80% glycerol for 12 h to render them transparent. Photographs were taken with a Nikon DMX1200 digital camera (Nikon, Japan) mounted on a Nikon SMZ1500 stereoscope (Nikon, Japan) and automatically stacked using Helicon Focus 3.2 (Helicon Software, USA). The median distance between the bases of socii of male genitalia (marked in Fig. 4) is a useful morphometric character, helpful to distinguish species (Koiwaya 1993); this distance, therefore, was measured to 0.2 mm precision for all dissected male genitalia. After observation and photography, all parts of the genitalia were fixed on a card with water soluble white glue and pinned with the specimen. The terminology of male and female genitalia followed Hu et al. (2018).

Molecular work
For specimens used in molecular work, one or two legs (except forelegs) on the same side were extracted, homogenised in protease buffer containing 100 μL STE (10 mmol/L Tris-HCl, 1 mmol/L EDTA, 100 mmol/L NaCl, pH = 8.0) and 2 μL Proteinase K (20 mg/mL) (O'Neill et al. 1992). Homogenised samples were treated at 37 °C incubation for 15 min to rehydrate the tissue and then at 95 °C incubation for 10 min to lyse the tissue. The supernatant was recovered through centrifugation at 6,000g and used directly as DNA template in polymerase chain reactions (PCR).

Phylogenetic analyses
Raw sequences were proofread and aligned using Clustal W (Thompson et al. 1994) in BioEdit 7.0.9 (Hall 1999), and any sequence containing double peaks in the chromatograms was strictly excluded. The product sequences were checked by MEGABLAST against the genomic references and nucleotide collection in NCBI. Amino acid translation was realised with the invertebrate mitochondrial criterion in MEGA 6.0 (Tamura et al. 2013) to detect possible Numts (nuclear copies of mtDNA fragments). A search for nonsynonymous mutations, in-frame stop codons, and indels was carried out to further minimise the existence of cryptic Numts (Song et al. 2008;Bertheau et al. 2011). Sequences used in the present study were listed in Table 1.
The phylogeny was reconstructed using Bayesian Inference (BI) method as implemented in MrBayes 3.2.6 ( Ronquist et al. 2012), with the most appropriate partition scheme recovered by PartitionFinder 2.1.1 (Lanfear et al. 2017) using the unlinked branch lengths and the greedy algorithm. We used the partitioning scheme and among-site rate variation suggested by PartitionFinder, but instead of selecting one substitution model a priori, we used reversible-jump Markov Chain Monte Carlo (rj-MCMC) to allow sampling across the entire substitution rate model space (Huelsenbeck et al. 2004). BI analyses consisted of two independent runs, each with eight rj-MCMC running for 10 million generations (sampled every 1000 th generation) to calculate the clade posterior probabilities (PP).
As in Hu et al. (2018), we used an individual of Iphiclides podalirius (Linnaeus, 1758) and of Lamproptera meges (Zinken, 1831), because we found that tree topology and node support were improved when using outgroup species that branched before Pazala. We reconstructed the phylogeny for a dataset containing the currently recognised species of Pazala (sensu Hu et al. 2018) in an attempt to obtain an overall phylogenetic framework for the subgenus and to produce phylogenetic relationships among the focal taxa in the present study.
Phyletic properties of each taxon in the alebion and tamerlanus groups were assessed using an online tool, Monophylizer (Mutanen et al. 2016; http://monophylizer.naturalis.nl/). Taxa identified as monophyletic were treated as good species or subspecies, while those identified as paraphyletic were further analysed using morphological characters and geographical ranges.

Molecular dating
To estimate divergence times and infer their 95% credibility intervals (CI), we performed Bayesian relaxed-clock analyses using MrBayes. For these analyses, we relied on the partitioning scheme and all the MrBayes settings as determined above. Dating analyses were realized with two independent runs for 20 million generations with sampling tree every 2,000 generations. We used the autocorrelated clock model (noted TK02 in MrBayes) for two reasons. First, the autocorrelated model is more appropriate for our dataset because the rate along a given branch is more similar to its parent branch than a branch chosen at random, though autocorrelation models differ in the degree to which they restrict rate variation between parent and daughter branches (Thorne et al. 1998;Thorne & Kishino 2002). Second, Lepage et al. (2007) and Rehm et al. (2011) showed that the autocorrelated clock model generally offers the best fit, as compared to the uncorrelated model and a strict molecular clock model. Unlike previous dating analyses on Pazala (Hu et al. , 2019, which relied on the TK02 clock model, better dating results were obtained in the present study using the IGR clock model. Calibration priors are based on the time-calibrated tree of Papilionidae (Condamine et al. 2012). We set four secondary calibrations using a (conservative) uniform prior with bounded by the minimum and maximum ages of the 95% CI of the divergence times (a normal prior is not recommended, Schenk 2016). We could not use fossils in this study because the three fossils do not belong to the subfamily Papilioninae (Condamine et al. 2012). We calibrated the following nodes: (i) the root of the tree (crown of Leptocircini) set between 27.6 and 43.4 million years ago (Ma); (ii) the crown between Iphiclides and Lamproptera set between 20.8 and 35.5 Ma; (iii) the crown of the genus Graphium set between 21.2 and 35.7 Ma; and (iv) the crown of the subgenus Graphium set between 14.4 and 29.7 Ma. Following Hu et al. (2018Hu et al. ( , 2019, Graphium agamemnon (KC970100) and G. sarpedon (KF401961) were added to the molecular dataset to better calibrate the molecular clock.
For all Bayesian runs (phylogenies and dating), convergence was ensured by checking average deviation of split frequencies (ADSF), potential scale reduction factor (PSRF) values, effective sample size (ESS) of all parameters, and by plotting log-likelihood of samples against number of generations in Tracer 1.7 (Rambaut et al. 2018). To reach good convergence, the runs must have values of ADSF approaching zero, PSRF close to 1.00 and ESS above 80 · Zootaxa 4759 (1) © 2020 Magnolia Press 200. Bayesian consensus trees were obtained using the 25% burn-in criterion , and the remaining samples were used to generate a 50% majority rule consensus tree.
All phylogenetic and dating analyses were performed on the computer cluster CIPRES Science Gateway (Miller et al. 2015), using BEAGLE ) with default parameters.

Phylogenetics of the alebion & tamerlanus groups
Bayesian phylogenetic analyses converged well as indicated by ADSF close to 0, PSRF equal to 1, and ESS >> 200 for all parameters. The corresponding phylogeny of the alebion and tamerlanus groups showed that species were well defined as monophyletic with high (if not maximal) posterior probabilities ( Figure 1). Confirming previous works , we found G. (P.) alebion as the sister species to all Pazala groups, including the tamerlanus group containing G. (P.) parus and G. (P.) tamerlanus. In the G. (P.) tamerlanus branch, two subspecies, namely ssp. tamerlanus and ssp. kansuensis, are identified. The Monophylizer analysis identified all three species in the two groups as monophyletic, while G. (P.) tamerlanus kansuensis entangled (but not mixed) with G. (P.) tamerlanus tamerlanus forming a paraphyletic clade in the tree ( Table 2). The phylogenetic structure and Monophylizer assessment of the two groups indicate the following two taxonomic results: (i) all three species are distinct species, differing from each other in the tree; (ii) G. (P.) tamerlanus kansuensis is genetically rather close to the nominate subspecies of G. (P.) tamerlanus, but is morphologically a good subspecies.
Morphological examination, detailed in the revision section below, supported the species level phylogenetic analysis particularly in differences of both male and female genitalia.   Diagnostic characters: Small in size, forewing length: male 31.0-33.5 mm (mean = 32.1 ± 1.0 mm, n = 4), female 33.5-35.0 mm (mean = 34.1 ± 0.6 mm, n = 4). Both wings distinctively narrow and elongate, ground colour with a light ochreous hue. Forewing the 1 st , 2 nd , 8 th , 9 th black bands and the terminal (10 th ) black band all reach tornal margin in both sexes; area between the 8 th and 9 th bands almost devoid of dark scales; the 7 th band not displaced inward in cell R 4 ; veins CuA 2 to M 2 stained with black distally. Hindwing discal band broadened towards costa; a whitish small patch at the base of tail in cell M 3 ; tornal yellow spots large and undivided on both sides.
Nomenclatorial note: The type specimen in BMNH was labelled as 'Lectotype' by Campbell Smith with the intention of designating it as such in a subsequent revision, similar to the type of Papilio glycerion Gray, 1831. A literature search confirmed that this specimen has not actually been formally designated as the lectotype, and since Gray did not state the number of specimens when he described Papilio alebion the specimen must be treated as a syntype. Since there is no clear reason requiring lectotype designation in this case, we refrain from doing so in this publication.
Distribution: E. China (Zhejiang, Jiangxi, Jiangsu, Anhui, Hunan). Phenology: Univoltine in mid-March to early April. Host plant: Lindera rubronervia (Lauraceae) is reported in Nanjing, Jiangsu . Host plant usage in other parts of its distribution range needs further study.
Male genitalia (Figure 4): Five male genitalia in total were dissected, and the general characters were consistent. Highly sclerotized. Ring slightly wavy in the upper half; saccus rather reduced; socius acute and smooth, distance between the base of socii 0.48-0.52 mm (mean = 0.50 ± 0.01 mm, n = 5). valve short, oval in general, mostly dark in colour except for the base (unique in subgenus Pazala); dorsal terminal harpe triangulate, edge serrate with the base widely separated apart; the medial harpe long and nearly straight, the dorsal projection almost flat, broader at the base and gradually narrowed into a pointed tip; no tooth in the middle of the medial harpe. Aedeagus shorter than the other species, almost straight. Juxta very small and narrow, highly sclerotized without hair, the base not directly associated with the base of valves. REvISION   Female genitalia ( Figure 5): In total, three females were available for dissection, and the characters were consistent. Lamella postvaginalis very reduced; lamella antevaginalis broad horizontally; ostial lobe heavily sclerotized, broad at the base and gradually narrowed into a blunt tip in lateral view, while the posterior margin smoothly wavy without any indentation or bifurcation in ventral view.

Graphium (Pazala) parus (Nicéville, 1900) (Figure 6)
Papilio parus Nicéville, 1900  Diagnostic characters: Larger than G. (P.) alebion, forewing length: male 33.5-40.5 mm (mean=37.6 ± 1.4 mm, n = 83), female 39.5-42.0 mm (mean = 40.5 ± 1.3 mm, n = 3). Both wings broader, ground colour dull creamy white with a slight greyish tinge; all black markings on both wings rather thick and prominent. Forewing the 1 st , 2 nd , 8 th , 9 th black bands and the terminal (10 th ) black band all reach tornal margin in both sexes; area between the 8 th and 9 th bands distinctively filled by dark scales (the extent varies among individuals but without geographical association) in male, but not in the female (only indicated near the apex); the 7 th band not displaced inward in cell R 4 ; veins CuA 2 to M 2 stained with black distally. Hindwing discal band not broadened towards costa; no whitish small patch at the base of tail in cell M 3 ; tornal yellow spots much smaller and divided on both sides.
Host plants: Shao-Ji Hu observed females in ovipositing posture around bushes of Litsea chuii var. likiangensis (Lauraceae) in Yulong Xueshan, N.W. Yunnan, but no eggs were collected for rearing to confirm whether this Litsea is its host plant.
Male genitalia (Figure 7): In total, 20 male genitalia of specimens collected from Yunnan and Sichuan were dissected, and the general characters were consistent. Highly sclerotized. Ring slightly wavy in the upper half; saccus small but moderately sclerotized; socius toothed laterally, distance between the base of socii 0.58-0.70 mm (mean = 0.64 ± 0.05 mm, n = 20). valve short, oval in general, dorsal terminal harpe long pear-shaped, edge serrate with the base separated; the medial harpe long and slightly curved, the dorsal projection bayonet-shaped with pointed or toothed tip; no tooth in the middle of the medial harpe. Aedeagus long, strongly curved ventrally. Juxta long, weakly sclerotized with hairy membrane on both sides.

Diagnostic characters:
The largest of the three species, both wings broad, ground colour pale white; all black markings on both wings thinner. Forewing the 1 st , 2 nd , 8 th black bands all reach tornal margin in both sexes, while the 9 th black band tends to be reduced near the tornus, and the terminal (10 th ) black band usually only reaches or just crosses vein CuA 2 ; dark scales between the 8 th and 9 th bands only indicated near the apex; the 7 th band not displaced inward in cell R 4 ; veins CuA 2 to M 2 only faintly stained with black distally. Hindwing discal band not broadened towards costa; no whitish small patch at the base of tail in cell M 3 ; tornal yellow spots much smaller and divided on both sides.
Male genitalia (Figure 10): In total, 10 male genitalia were dissected for the two known subspecies, namely ssp. tamerlanus and ssp. kansuensis, and the general characters were consistent. Highly sclerotized. Ring slightly wavy in the upper half; saccus small but moderately sclerotized; socius toothed laterally, distance between the base of socii 0.40-0.60 mm (mean = 0.49 ± 0.08 mm, n = 10). valve short, oval in general, dorsal terminal harpe short, pear-shaped with both lower angles more acute, edge serrate with the base connected; the medial harpe long and slightly curved, the dorsal projection bayonet-shaped with pointed or toothed tip; no tooth in the middle of the medial harpe. Juxta weakly sclerotized with hairy membrane on both sides. REvISION (Figure 11): In total, two females of ssp. tamerlanus and a female of ssp. kansuensis were available for dissection, and the overall characters were consistent. Lamella postvaginalis small; lamella antevaginalis broad horizontally, covered with sclerotized wrinkles; ostial lobe heavily sclerotized, broad at the base and gradually narrowed into a triangular tip in lateral view, the posterior margin smooth in ventral view in two females of ssp. tamerlanus, and only very shallowly indented into "W"-shape in the only female of ssp. kansuensis.    Wang, Chen & Wang, 1990, Butt. Fauna Henan: 7, pl. 9, f. 12. [Nomen nudum, attributed to Oberthür] Diagnostic characters: Smaller than the nominate subspecies, forewing length: male 33.5-39.0 mm (mean = 37.3 ± 1.6 mm, n = 17), female 39.0-40.0 mm (mean = 39.7 ± 0.6 mm, n = 3). Both wings narrower and slightly elongate, the 9 th black band of forewing reduced even before vein CuA 2 in both sexes, the subterminal double black bands obviously close to each other.

Female genitalia
Distribution: Confined to the Qinling Mountains in S.E. Gansu, southern Shaanxi and western Henan provinces, China.
Phenology: Specimen records indicate the flight period mainly lasts from April to June, but also as late as August (syntypes). Whether this species is univoltine or bivoltine requires further investigation.
Host plant: Unknown. Probably a plant belonging to the family Lauraceae.

Discussion
The alebion group is relatively old in phylogenetic position, diverging before all other species in subgenus Pazala. Unlike other groups that possess subspecies and cryptic taxa throughout their distribution range (Racheli & Cotton 2009;Hu et al. 2018;Hu et al. 2019), there is no taxon diversity within the alebion group. G. (P.) alebion is the only species in this group, and our analyses did not identify any new taxa. Although the taxa within the tamerlanus group diverged later than those of the mandarinus, eurous, and mullah groups, it also lacks low-altitude sister taxa compared to the mandarinus and eurous groups (Racheli & Cotton 2009;Hu et al. 2018;Hu et al. 2019), indicating slow historical radiation for this group. G. (P.) tamerlanus is the only species with subspecies in this study, and molecular evidence inferred that the divergence between the two subspecies separated by the Qinling Mountains is quite recent (Table 3; Figure 2). Since the Qinling Mountains arose in a significantly old geological time (Meng 2017), the authors tend to believe the split of the two subspecies might more likely be attributed to climate change in the Pleistocene as stated by Hu et al. (2018) rather than tectonic shifts. Taxonomic and distribution confusions have long been coupled with each other among the three focal species of this study. Mell (1938) (Oberthür)', which is actually a pale example of G.

REvISION OF PAZALA
Zootaxa 4759 (1) © 2020 Magnolia Press · 91 (P.) parus; the same specimen was again illustrated in Lee (1995: 52, f. 68). Racheli & Cotton (2009) stated under the taxonomic note for G. (P.) tamerlanus: "In ZFNIK [sic = ZFMK], in the Höne Collection, there are typical specimens of tamerlanus from A-tun-tse [Mekong river north of Yanmen, N Yunnan]." However, a careful re-examination of the photographed specimens from the Höne Collection of the ZFMK showed that all the females were G. (P.) parus rather than G. (P.) tamerlanus (Figure 13). Similarly, on examination in 2006, we found that the three 'tamerlanus' drawers in the collection of BMNH also contained a mixture of G. (P.) parus and G. (P.) tamerlanus. Campbell Smith curated the BMNH specimens in 2003, labelling them 'f. tamerlanus' and 'f. incerta'. He also determined a male of G. (P.) parus from N. Kachin State, Myanmar, as 'Graphium t. tamerlanus f. parus'. Possibly Smith had not seen Koiwaya (1993) in which he recognised Pazala incerta [= G. (P.) parus] as a separate species to tamerlanus. Racheli & Cotton (2009) realised that parus was the senior name for the same taxon as incertus Bang-Haas, thus confirming the valid species name. Specimens sold in the west under the name 'Pazala alebion' from Sichuan in recent years have all proven to be either G. (P.) tamerlanus or G. (P.) parus. This may partly be due to confusion in the works of D'Abrera (1982,1990), which many collectors and commercial suppliers have used for identification purposes. In his 1982 book on Oriental butterflies, D'Abrera treated G. (Pazala) mullah chungianus from Taiwan as 'Pazala alebion chungiyanus [sic]' and in the Holarctic volume (1990) he illustrated tamerlanus as a subspecies of 'Pazala alebion' and pictured a male of Graphium (Pazala) mullah alongside an obviously different specimen of true alebion, both captioned as 'P. alebion alebion'. Possibly D'Abrera was also confused by the Shirôzu's (1961) description of Iphiclides alebion tayal, which is a junior synonym of G. (Pazala) mullah chungianus (Murayama, 1961). Murayama originally described chungianus as a species in genus Iphiclides two months before Shirôzu published his subspecies name.
To date, all reliable records of G. (P.) alebion are still confined to the lower Yangtze watershed in E. China ( Figure 14); no specimens of G. (P.) alebion have been found in C. China or westward areas approaching the two aforementioned species. Racheli & Cotton (2009) illustrated a female supposedly from Taibai Shan, Shaanxi (photo obtained by the editor, G. C. Bozano, from an unverified source). However, Prof. Yu-Fei Li (Xi'an, China) has studied butterflies of Taibai Shan for over 20 years and stated (pers. comm.) that he had never seen G. (P.) alebion there in his lifetime. The widely ranging distribution for this species recorded in Chou (1994) including most of China and India must comprise many misidentifications. Wu (2001) similarly includes Sichuan and even Yunnan in the distribution of alebion. Further surveys in C. China in late February to March are required to finally elucidate the western limit of this species. We think that such confusions were mainly caused by the similar wing pattern and male genitalic structure of the three species, especially the single, non-bifurcated hindwing discal band, which differs from all other Pazala species. However, the micro characters on both wings and the terminal process of male genitalia can be used to separate the three species effectively. Our study confirmed that the ostial lobes in female genitalia of the three species are totally different, demonstrating that they are three distinct species. This is also supported by the Bayesian phylogenetic tree and the estimates of divergence times showing three clades (Figure 1). Racheli & Cotton (2009) speculated a closer relationship between G. (P.) alebion and G. (P.) mullah than the tamerlanus group, based on the large undivided tornal yellow spots and forwardly expanded hindwing discal band. Our analysis and a previous study (Figure 1; Hu et al. 2018) showed that G. (P.) mullah was more distant than the tamerlanus group, which does not possess such characters. We think the two typical characters might represent the ancestral state of G. (P.) alebion which were lost in the course of divergence into the tamerlanus group and regained by G. (P.) mullah.
Our field survey and collection covering most altiplano and mountains of N. and N.W. Yunnan over a decade never found G. (P.) tamerlanus. All reliable records of G. (P.) tamerlanus extend from the western margin of the Sichuan Basin northeastwardly to the southwest corner of the Qinling Mountains. In comparison, the distribution range of G. (P.) parus occupies the entire montane area in the upper Irrawaddy, Salween, Mekong, and Yangtze watersheds in W. and N.W. Yunnan, and extends northeastwardly to the western margin of the Sichuan Basin, where G. (P.) tamerlanus is sympatric (Figure 14). The range of both species may be slightly wider than recorded, but based on the current data, it is unlikely that G. (P.) tamerlanus would reach Yunnan across the Yangtze valley.