Haydon Warren-Gash Et Al.: Mylothris Phylogeny

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Nota Lepi. 43 2020: 1–14 | DOI 10.3897/nl.43.46354 Research Article

Systematics and evolution of the African butterfly genus Mylothris (Lepidoptera, Pieridae)

Haydon Warren-Gash1, Kwaku Aduse-Poku2, Leidys Murillo-Ramos3,4, Niklas Wahlberg3

1 98 Overstrand Mansions, Prince of Wales Drive, London SW11 4EU, UK 2 Biology Department, University of Richmond, 138 UR Drive, Richmond, VA 23173, USA 3 Department of Biology, Sölvegatan 37, Lund University, 223 62 Lund, Sweden 4 Grupo Biología Evolutiva, Department of Biology, Universidad de Sucre, Sincelejo, Sucre, Colombia http://zoobank.org/CB00DD10-8657-4283-93BA-0FEFDEB922A7

Received 6 September 2019; accepted 6 January 2020; published: 11 February 2020 Subject Editor: Roger Vila.

Abstract. We study the systematics and evolutionary history of the Afrotropical butterfly genus Mylothris (Lepidoptera: Pieridae) based on six gene regions (COI, EF1a, GAPDH, MDH, RpS5 and wingless). We find that the genus can be placed into five species groups, termed thejacksoni , elodina, rhodope, agathina and hilara groups. Within these species groups, we find that many species show very little genetic differentiation based on the markers we sequenced, suggesting they have undergone rapid and recent speciation. Based on secondary calibrations, we estimate the age of the crown group of Mylothris to be about 16 million years old, but that many of the species level divergences have happened in the Pleistocene. We infer that the clade has its origin in the forests of the Eastern part of Central Africa, and has spread out from there to other regions of Africa.

Introduction Studies on the systematics of Afrotropical butterflies using molecular data have been increasing in number in the past decade (Kodandaramaiah and Wahlberg 2007; Aduse-Poku et al. 2009; Nazari et al. 2011; Price et al. 2011; van Velzen et al. 2013; Aduse-Poku et al. 2017), giving us a better understanding of their evolutionary histories in one of the diversity hotspots of the planet. Many genera are endemic to the continent, although there are clear connections to the Oriental region (Kodandaramaiah and Wahlberg 2007; Aduse-Poku et al. 2009; Aduse-Poku et al. 2015; Sahoo et al. 2018; Toussaint et al. 2019) and even to the Neotropical region (Silva Brandão et al. 2008). Genera in the family Pieridae have not been the focus of many studies to date. So far only two pierid genera have been studied using molecular approaches in any detail, Colotis Hübner, 1819 (Nazari et al. 2011) and Pseudopontia Plötz, 1870 (Mitter et al. 2011). Here we investigate the systematics and evolutionary history of the Afrotropical pierid genus Mylothris Hübner, 1819. The range of Mylothris includes most of the African continent south of the Sahara, extending to nearby islands such as Madagascar and the Comoros in the east, and to Bioko and São Tomé e Príncipe in the west. A single species flies alongside other pierid species belonging to the Afrotrop- ical fauna in south west Arabia. It is speciose: a revision of the genus by one of the authors (HWG) is in progress and will list 105 species, a number of them new, many of them in turn divided into several subspecies (Warren-Gash in press). 2 Haydon Warren-Gash et al.: Mylothris phylogeny

A distinguishing feature of the genus, as with the related Catasticta Butler, 1870 in the Neotrop- ics (Braby and Nishida 2010) and Delias Hübner, 1819 in the Australasian region (Braby 2006), is that the hostplants, with one known exception, are members of parasitic mistletoes belonging to the family Loranthaceae. This in turn determines where and how plentifully Mylothris species are found. Their foodplants are arboreal epiphytes and so they are insects of the canopies – unlike most Afrotropical pierids, which breed on low-growing shrubs such as Capparis Linnaeus, 1758, and many of which occur in more open, drier biotopes. Thus Mylothris species diversity is richest in the main African forest belt, with some species spreading into the forest-savannah mosaic and anthropogenic biomes including suburban gardens. Two other consequences follow. The first is that, since Loranthaceae occur most frequently near the crown of the tree, many of the tropical forest species fly high, and some of them are seldom seen, accentuating their rarity in institutional collections. Secondly, they are particularly vulnerable to primary forest degradation. Typically, Mylothris eggs are laid in a group on the underside of a leaf of the hostplant, and the larvae when they hatch are gregarious. The length of the lifecycle depends on the species and the biome or region. In southern Africa they follow the seasons in line with their foodplant, as they do in colder (usually montane) climates further north. In less seasonal biotopes, adults can be on the wing at any time of year (Warren-Gash in press). In appearance Mylothris are a remarkably homogeneous group. In adults of the M. jacksoni species group, the hindwing is yellow and the forewing usually mostly white. The other clades typically show some orange (sometimes yellow) at the base of the forewing on one or both surfaces becoming white distally, with a greater or lesser black distal margin. There are exceptions, but they are similar enough to have baffled taxonomists over the years, leading to groups of species being lumped together under a single name in some cases and, no less frequently, names erected to describe what are no more than individual variations. If that were not enough, cryptic rings of different species that in adult morphology are externally almost indistinguishable, can be found flying together, most frequently in the two main areas of diversity: the Albertine Rift and central Cameroon (Warren-Gash in press). The forthcoming revision (Warren-Gash in press) will look at all these issues in more detail based on morphological characters. Here we aim to investigate whether molecular data can shed light on species delimitations and their relationships. As yet undescribed species are here named nsp1 to nsp4. They will be formally described in Warren-Gash (in press).

Material and methods We attempted to sample as many species as possible, and for selected species, as many populations as possible. We included 52 out of 105 species and a total of 235 individuals in our analyses (see Suppl. material 1: Table S1). Most specimens come from the private collection of HWG, with a few specimens taken from the collections of the African Butterfly Research Institute in Nairobi, Kenya. The specimens are part of a large taxonomic revision of Mylothris (Warren-Gash in press). In addition we included 23 outgroups from the subtribe Aporiina, taken from a previous publication (Wahlberg et al. 2014). DNA was extracted from 2–3 legs of dried museum specimens using the Nucleospin Tissue Kit (Macherey-Nagel, Düren, Germany). From these specimens, we sequenced the mitochondrial COI gene and up to 5 nuclear genes (EF1a, GAPDH, MDH, RpS5 and wingless). Primers and laboratory protocols followed Wahlberg and Wheat (2008) in all cases. Successful PCR products were cleaned Nota Lepi. 43: 1–14 3 enzymatically with exonuclease and FastAP (Thermo Fisher Scientific, Hampton, NH, USA) and sent to Macrogen Europe (Amsterdam, the Netherlands) for Sanger sequencing. The resulting chromato- grams were examined by eye using BioEdit (Hall 1999). All six genes are protein-coding, thus align- ments were trivial. The aligned sequences were curated and managed in VoSeq (Peña and Malm 2012). Phylogenetic analyses were carried out using IQ-TREE 1.6.10 (Nguyen et al. 2015) in a maximum likelihood framework. The full dataset was analysed. The data were partitioned by gene and analysed with the partition finding (Chernomor et al. 2016) and model finding (Kalyaanamoorthy et al. 2017) algorithms of IQ-TREE (using the command MFP+MERGE). Robustness of the results were assessed using UFBoot2 (Hoang et al. 2018) and a SH-like approximate likelihood ratio test (Guindon et al. 2010). Analyses were run on the CIPRES server (Miller et al. 2010). Timing of divergence was estimated using a reduced dataset. One specimen per species with the most DNA sequence data available was chosen for this analysis. The dataset was analysed using BEAST v1.8.3 (Drummond et al. 2012), partitioned according to the results of the IQ-TREE analysis. Since BEAST does not offer the possibility to use the TIM2 model, the model for COI was set to the GTR+G model, the other two partitions were assigned models according to the IQ-TREE results (see below). The nucleotide models and clock models were unlinked between partitions. The tree prior was set to the birth-death model and a lognormal relaxed clock was implemented. Monophyly of Aporiina was enforced. Four secondary calibration points were taken from Chazot et al. (2019) and were modeled with a normal prior. The root was set to 42 million years ago (Mya) ± 5 million years (My), the crown age of Aporiina to 36 Mya ± 4 My, the most common recent ancestor of Mylothris and Aporia Hübner, 1819 to 28 Mya ± 3.5 My, and the most recent common ancestor of Catasticta and Melete Swainson, 1831 to 20 Mya ± 3 My. The analyses were run four times independently at 10 million generations per run sampling at 10,000 generation intervals. Convergence was examined using Tracer v1.7 (Rambaut et al. 2014). The four runs were combined after a burnin of 1 million generations and a summary tree was generated using the maximum a posteriori tree and mean estimated times of divergence. The historical biogeography of the group was inferred using the R package BioGeoBEARS v1.1.1 (Matzke 2014). The chronogram resulting from the BEAST analysis (see results section) with the outgroups pruned, was used for this analysis. Owing to the open conceptual debate on model comparisons in BioGeoBEARS (Ree and Sanmartín 2018), our ancestral range analysis were performed under the dispersal – extinction – cladogenesis (DEC) model (Ree et al. 2005; Ree and Smith 2008). DEC is a likelihood-based procedure that models range evolution as a discrete-valued process, specifying instantaneous transition rates between geographical ranges along the branches of a phylogenetic tree. Consequently, DEC requires each sampled taxon be assigned with one or more geographic range(s), reflecting its present distributional range. The geographic distributions of Mylothris were extrapolated from the literature and the degree of endemism of local species, which is highest in West Africa and Madagascar. The following regions were used in the analysis: A, West Africa; B, West Central Africa; C, East Central Africa; D, East Africa; E, South Africa; F, Malagasy Region (see inserted map in Figure 6 of Results section). B and C are closer than the others, corresponding roughly to the central forest belt, but with two main centres of endemism: the Cameroon highlands and the montane zone just west of the Albertine Rift. In all dispersal rate scaler matrices, dispersal events between adjacent areas were not scaled (dispersal d = 1.0). A scaler of 0.5 was applied for dispersal events between areas separated by an ocean barrier. A scaler of 0.25 was applied for dispersal events between areas separated by a third region and a water barrier (see Suppl. material 2: Table S2). The maximum number of areas per ancestral state was fixed to four. 4 Haydon Warren-Gash et al.: Mylothris phylogeny

Results The dataset consisted of 5168 aligned base pairs. The IQ-TREE partition finding analysis combined EF1a, GAPDH, MDH, and RpS5 into one partition and assigned the GTR+I+G model to it. COI was kept as its own partition with TIM2+I+G model assigned to it, as was wingless with the K2P+G model. We find Mylothris to be a well supported monophyletic group (UFB = 100, SH-like = 100), that is sister to a clade of Neotropical and Palaearctic/Oriental taxa (Figure 1). We identify five major clades that are strongly supported (the exception being the hilara clade with UFB = 87, SH- like = 90): the jacksoni, elodina, rhodope, agathina and hilara clades. We find the jacksoni clade to be sister to the rest of Mylothris, with elodina being the next lineage to diverge, followed by the rhodope clade, and finally theagathina and hilara clades being sister to each other. Relationships within the jacksoni clade show four lineages (Figure 2), with M. ducarmei Hecq, 2001 being sister to the rest, followed by M. crawshayi Butler, 1896 and finallyM. trimenia Butler,

Delias clade Aporia Catasticta clade 69.4/55 100/100 Aporiina jacksoni group 27.2/55 99.5/100 elodina 100/100 group 100/100

rhodope group 100/100 85.3/79

100/100 agathina group 100/100

91.4/79

hilara group 89.6/87

0.04

Figure 1. Relationships of the five species groups inMylothris based on a maximum likelihood analysis of 5 gene regions. Numbers below branches are branch support values (SH-like / Ultrafast Bootstrap). Nota Lepi. 43: 1–14 5

1869 sister to a clade comprising five species with unresolved relationships. These five species,M. jacksoni Sharpe, 1891 M. sagala, Grose-Smith, 1886 M. knutssoni Aurivillius, 1891 M. nagichota Talbot, 1944 and M. seminigra d’Abrera, 1980 show very little genetic differentiation. Only M. elodina Talbot, 1944 is found in the elodina clade (Figure 2). The eight individuals we sequenced show very little genetic differentiation, but a relatively long branch leads to them. The position of the lineage is not highly supported (UFB = 79, SH-like = 85.3), and there is a possibility that further data might place it as the sister to the rest of Mylothris. The rhodope clade comprises 14 sampled species (Figure 3), which are genetically very close to each other. Indeed, M. kiwuensis Grunberg, 1910, M. knoopi Hecq, 2005, M. nsp2 and M. jaopura Karsch, 1893 are genetically identical at all the sequenced loci. Mylothris uniformis Talbot, 1944 and M. nsp3 are also intermixed and genetically extremely close to each other. Other species are reciprocally monophyletic, but genetically very close to their sister species, such as M. nsp4 + M. rhodope Fabricius, 1775 and M. latimargo Joicey & Talbot, 1921+ M. zairiensis Berger, 1981.

HWG1_001 Mylothris ducarmei 100/100 HWG1_002 Mylothris ducarmei 99.5/100 HWG1_175 Mylothris crawshayi 99.4/100 HWG1_176 Mylothris crawshayi HWG1_162 Mylothris trimenia 91.5/91 100/100 HWG1_163 Mylothris trimenia HWG1_164 Mylothris trimenia 96.4/99 HWG1_007 Mylothris jacksoni HWG1_004 Mylothris jacksoni 100/100 HWG1_006 Mylothris jacksoni HWG1_005 Mylothris jacksoni HWG1_256 Mylothris jacksoni HWG1_261 Mylothris jacksoni HWG1_262 Mylothris knutsoni HWG1_008 Mylothris jacksoni HWG1_279 Mylothris jacksoni HWG1_180 Mylothris sagala HWG1_177 Mylothris seminigra HWG1_178 Mylothris seminigra 100/100 HWG1_011 Mylothris nagichota HWG1_009 Mylothris knutsoni HWG1_010 Mylothris knutsoni HWG1_003 Mylothris jacksoni ABRI_17_2370 Mylothris sagala ABRI_17_2372 Mylothris sagala ABRI_17_2373 Mylothris sagala ABRI_17_2369 Mylothris sagala ABRI_17_2371 Mylothris sagala ABRI_17_2374 Mylothris sagala HWG1_053 Mylothris elodina HWG1_274 Mylothris elodina 100/100 HWG1_054 Mylothris elodina HWG1_225 Mylothris elodina HWG1_223 Mylothris elodina HWG1_222 Mylothris elodina 85.3/79 HWG1_055 Mylothris elodina HWG1_221 Mylothris elodina

100/100 0.04 Figure 2. Relationships of sampled specimens within the jacksoni and elodina groups. Numbers below branches are branch support values (SH-like / Ultrafast Bootstrap). 6 Haydon Warren-Gash et al.: Mylothris phylogeny

HWG1_014 Mylothris croceus 100/100 HWG1_015 Mylothris croceus HWG1_226 Mylothris sophiae HWG1_049 Mylothris kiwuensis 100/83 HWG1_075 Mylothris nsp2 100/48 HWG1_087 Mylothris nsp2 HWG1_144 Mylothris jaopura HWG1_089 Mylothris nsp2 HWG1_143 Mylothris jaopura 100/100 HWG1_066 Mylothris knoopi HWG1_123 Mylothris knoopi 100/100 HWG1_051 Mylothris kiwuensis HWG1_067 Mylothris knoopi HWG1_050 Mylothris kiwuensis HWG1_252 Mylothris knoopi HWG1_086 Mylothris nsp2 HWG1_085 Mylothris nsp2 HWG1_091 Mylothris katera HWG1_093 Mylothris nsp2 HWG1_141 Mylothris jaopura 85.3/79 HWG1_048 Mylothris kiwuensis 46.2/65 HWG1_052 Mylothris kiwuensis HWG2_005 Mylothris kiwuensis HWG1_276 Mylothris knoopi HWG1_088 Mylothris nsp2 HWG1_224 Mylothris nsp2 HWG1_254 Mylothris knoopi HWG1_307 Mylothris knoopi HWG1_042 Mylothris yulei 99.7/99 HWG1_044 Mylothris yulei 97.1/88 HWG1_083 Mylothris uniformis HWG1_069 Mylothris uniformis 99.2/95 HWG1_217 Mylothris uniformis HWG1_270 Mylothris uniformis HWG1_302 Mylothris nsp3 HWG1_079 Mylothris uniformis HWG1_253 Mylothris nsp3 52.9/63 HWG1_036 Mylothris hecqui 100/100 HWG1_037 Mylothris hecqui HWG1_072 Mylothris nsp4 79/66 HWG2_010 Mylothris nsp4 100/100 HWG1_216 Mylothris nsp4 HWG1_239 Mylothris nsp4 HWG1_299 Mylothris nsp4 97.6/9198/87 HWG1_218 Mylothris nsp4 HWG1_295 Mylothris nsp4 HWG1_303 Mylothris rhodope HWG1_058 Mylothris rhodope 92.1/96 HWG1_206 Mylothris rhodope HWG1_205 Mylothris rhodope HWG1_245 Mylothris rhodope 88.2/80 HWG1_024 Mylothris rhodope HWG1_204 Mylothris rhodope HWG1_207 Mylothris rhodope HWG1_045 Mylothris latimargo 85.5/78 HWG1_046 Mylothris latimargo HWG1_269 Mylothris zairiensis HWG1_293 Mylothris zairiensis 99.3/99 HWG1_308 Mylothris zairiensis HWG1_309 Mylothris zairiensis HWG1_090 Mylothris zairiensis 91.8/88 HWG1_310 Mylothris zairiensis HWG1_294 Mylothris zairiensis HWG1_095 Mylothris zairiensis HWG1_071 Mylothris zairiensis HWG1_068 Mylothris zairiensis HWG1_240 Mylothris zairiensis HWG1_077 Mylothris zairiensis HWG1_297 Mylothris zairiensis 0.04

Figure 3. Relationships of sampled specimens within the rhodope group. Numbers below branches are branch support values (SH-like / Ultrafast Bootstrap). Nota Lepi. 43: 1–14 7

HWG1_019 Mylothris ochracea 24.3/54 HWG1_018 Mylothris ochracea HWG1_305 Mylothris ochracea HWG1_016 Mylothris marginea 100/100 94/89 98.9/94 HWG1_017 Mylothris marginea HWG1_171 Mylothris interposita HWG2_001 Mylothris aurantiaca 94.8/87 100/75 HWG1_273 Mylothris sulphurea HWG2_002 Mylothris aurantiaca 85.3/79 80.6/57 HWG1_219 Mylothris sulphurea HWG1_210 Mylothris sulphurea HWG1_227 Mylothris mavunda HWG1_287 Mylothris sulphurea HWG1_023 Mylothris sulphurea HWG2_012 Mylothris holochroma KA_17_0223_ABRI Mylothris aurantiaca HWG1_022 Mylothris sulphurea HWG2_003 Mylothris aurantiaca HWG2_013 Mylothris holochroma 100/100 HWG1_271 Mylothris sulphurea 100/100 HWG1_272 Mylothris sulphurea HWG2_004 Mylothris interposita KA_17_0222_ABRI Mylothris aurantica HWG1_021 Mylothris interposita HWG1_129 Mylothris sulphurea HWG1_202 Mylothris dimidiata 100/100 HWG1_101 Mylothris dimidiata HWG1_201 Mylothris dimidiata 93.2/93 HWG1_211 Mylothris asphodelus HWG1_128 Mylothris asphodelus 100/100 HWG2_009 Mylothris asphodelus HWG1_212 Mylothris asphodelus HWG1_249 Mylothris asphodelus HWG1_130 Mylothris asphodelus 61.6/62 HWG1_250 Mylothris asphodelus HWG1_251 Mylothris asphodelus HWG1_035 Mylothris canescens HWG1_039 Mylothris bernice 99.7/100 HWG1_040 Mylothris bernice 91.4/79 SC_01_M002 Mylothris bernice 99/94 HWG1_281 Mylothris bernice HWG1_173 Mylothris bernice HWG1_172 Mylothris bernice 82.9/84 HWG1_174 Mylothris bernice KA17_01NW Mylothris sp 98.1/100 HWG1_138 Mylothris ngaziya 98.9/92 HWG1_139 Mylothris ngaziya 98.1/89 DL14A_0078 Mylothris phileris 87.4/75 HWG2_021 Mylothris phileris HWG1_029 Mylothris agathina 83.3/63 HWG1_026 Mylothris chloris HWG1_025 Mylothris chloris

95.9/78 HWG1_030 Mylothris agathina HWG1_032 Mylothris agathina NP_99_T486 Mylothris agathina HWG1_028 Mylothris chloris HWG1_027 Mylothris chloris HWG1_169 Mylothris agathina HWG1_170 Mylothris agathina 0.04 89.6/87

Figure 4. Relationships of sampled specimens within the agathina group. Numbers below branches are branch support values (SH-like / Ultrafast Bootstrap). 8 Haydon Warren-Gash et al.: Mylothris phylogeny

100/100 85.3/79 HWG2_023 Mylothris similis 100/100 100/99 HWG1_244 Mylothris sjostedti 99.6/99 HWG1_033 Mylothris sjostedti 91.4/79 HWG1_034 Mylothris sjostedti HWG1_102 Mylothris poppea 100/100 HWG1_104 Mylothris poppea 89.6/87 HWG1_203 Mylothris poppea HWG1_098 Mylothris erlangeri 99.7/99 HWG1_099 Mylothris erlangeri HWG1_229 Mylothris ochrea 99/100 HWG2_016 Mylothris dubia HWG1_135 Mylothris carolinae HWG1_235 Mylothris dubia HWG1_096 Mylothris carolinae HWG1_236 Mylothris dubia HWG1_246 Mylothris dubia 100/100 KA_17_0546_ABRI Mylothris rueppellii HWG2_015 Mylothris dubia HWG1_124 Mylothris dubia HWG1_208 Mylothris dubia HWG1_117 Mylothris dubia HWG1_277 Mylothris dubia HWG1_267 Mylothris dubia 87/60 HWG1_233 Mylothris dubia HWG2_006 Mylothris dubia HWG1_214 Mylothris carolinae HWG1_242 Mylothris carolinae HWG1_137 Mylothris carolinae HWG1_134 Mylothris carolinae HWG1_133 Mylothris carolinae HWG1_136 Mylothris carolinae HWG1_237 Mylothris dubia HWG1_241 Mylothris continua HWG1_119 Mylothris winstoni HWG1_291 Mylothris carolinae HWG2_019 Mylothris maxima HWG1_215 Mylothris carolinae HWG2_007 Mylothris zairiensis HWG1_230 Mylothris carolinae KA_17_0220_ABRI Mylothris furvus KA_17_0221_ABRI Mylothris furvus HWG1_234 Mylothris continua HWG1_168 Mylothris rueppellii HWG1_120 Mylothris winstoni HWG1_165 Mylothris rueppellii HWG1_166 Mylothris rueppellii HWG1_213 Mylothris maxima HWG1_132 Mylothris maxima HWG1_232 Mylothris carolinae HWG1_126 Mylothris carolinae HWG1_275 Mylothris maxima HWG1_122 Mylothris maxima HWG1_248 Mylothris maxima HWG2_008 Mylothris continua HWG1_278 Mylothris maxima HWG1_301 Mylothris hilara HWG1_238 Mylothris dubia HWG1_285 Mylothris septentionalis HWG1_247 Mylothris dubia HWG2_018 Mylothris maxima HWG1_268 Mylothris continua HWG1_284 Mylothris septentionalis HWG1_282 Mylothris septentionalis HWG1_283 Mylothris septentionalis HWG1_231 Mylothris carolinae HWG1_243 Mylothris continua HWG1_108 Mylothris nsp1 0.04 HWG1_110 Mylothris nsp1 HWG1_103 Mylothris nsp1 HWG1_109 Mylothris nsp1 Figure 5. Relationships of sampled specimens within the hilara group. Numbers below branches are branch support values (SH-like / Ultrafast Bootstrap). Nota Lepi. 43: 1–14 9

ABCD EF HWG1_001 Mylothris ducarmei HWG1_176 Mylothris crawshayi HWG1_164 Mylothris trimenia HWG1_007 Mylothris jacksoni Jacksoni group HWG1_177 Mylothris seminigra HWG1_011 Mylothris nagichota HWG1_009 Mylothris knutsoni ABRI_17_2373 Mylothris sagala HWG1_225 Mylothris elodina Elodina group HWG1_014 Mylothris croceus HWG1_036 Mylothris hecqui HWG1_058 Mylothris rhodope HWG1_218 Mylothris nsp4 HWG1_045 Mylothris latimargo HWG1_068 Mylothris zairiensis HWG1_044 Mylothris yulei HWG1_302 Mylothris nsp3 Rhodope group HWG1_217 Mylothris uniformis HWG1_226 Mylothris sophiae HWG1_091 Mylothris katera HWG1_048 Mylothris kiwuensis HWG1_141 Mylothris jaopura HWG1_123 Mylothris knoopi HWG1_087 Mylothris nsp2 HWG1_033 Mylothris sjostedti HWG2_023 Mylothris similis HWG1_102 Mylothris poppea HWG1_117 Mylothris dubia HWG1_098 Mylothris erlangeri HWG1_137 Mylothris carolinae Hilara group HWG1_166 Mylothris rueppellii HWG1_213 Mylothris maxima HWG1_126 Mylothris carolinae HWG1_108 Mylothris nsp1 HWG1_282 Mylothris septentionalis HWG1_119 Mylothris winstoni Western Africa (A) HWG1_019 Mylothris ochracea HWG2_001 Mylothris aurantiaca West Central Africa (B) HWG1_017 Mylothris marginea HWG2_012 Mylothris holochroma East Central Africa (C) HWG1_021 Mylothris interposita Eastern Africa (D) HWG1_210 Mylothris sulphurea HWG1_227 Mylothris mavunda Southern Africa (E) HWG1_101 Mylothris dimidiata Agathina group Madgascar (F) HWG1_211 Mylothris asphodelus HWG1_035 Mylothris canescens HWG1_172 Mylothris bernice KA17_01NW Mylothris sp HWG1_138 Mylothris ngaziya DL14A_0078 Mylothris phileris NP_99_T486 Mylothris agathina HWG1_028 Mylothris chloris

15 10 5 0 Millions of years ago

Figure 6. Timing of divergence and historical biogeographic analyses of Mylothris. Inset shows the biogeo- graphical regions used for the latter.

The agathina clade has 16 sampled species in it, showing similar patterns of genetic differentiation as the previous clades (Figure 4). Mylothris agathina Cramer, 1779 and M. chloris Fabricius, 1775 are genetically indistinguishable (based on the markers used here), as are M. sulphurea Au- rivillius, 1895, M. holochroma Talbot, 1944, M. interposita Joicey & Talbot, 1921 and M. aurantiaca Rebel, 1914. The Madagascan M. phileris Boisduval, 1833 and Comoro Islands M. ngaziya Oberthur, 1888 are found to be closely related to the M. agathina/chloris clade. The hilara clade comprises 15 sampled species, and again similar patterns of little genetic differentiation are found among the species as in the other clades (Figure 5). The species M. sjostedti Aurivillius, 1895, M. similis Lathy, 1906, M. poppea Cramer, 1777 and M. erlangeri Pagenstecher, 1902 appear to be distinct from each other and the rest, while the rest of the species form a complex set of relationships with shared haplotypes and minor monophyletic groups (e.g. M. nsp1), which are very closely related genetically, even if morphological characters are clearly distinct. Our timing of divergence analysis suggests that the crown clade of Mylothris began diversify- ing around 16 Mya (Figure 6) after diverging from their sister group some 10 My earlier. The five major clades diverged from each other between 16 and 8 Mya, during the mid to late Miocene, but almost all species level divergences have happened in the Pleistocene. Our ancestral range analysis recovers an origin of Mylothris in the East Central Africa. All the five major clades of the group are also inferred to have been derived from ancestors distributed in the East Central Africa. The ancestors of the extant species in Madagascar and Comoros islands are estimated to have range expanded from mainland Africa only recently (~2 Mya). 10 Haydon Warren-Gash et al.: Mylothris phylogeny

Discussion Mylothris are a distinct and well-supported lineage of African pierids, not closely related to any other pierids on that continent. There are five clear species groups within the genus. Of the five groups, the rhodope, agathina and hilara species groups are more closely related to each other than to the other two groups. Based on our sampling of genes, it appears that the jacksoni group is sister to the rest, but with poor support. Indeed, in some preliminary analyses of our data, the elodina group came out as sister to the rest, and this may be the case with further gene and taxon sampling. The very different morphology of elodina, reflected also in the male genitalia, is a possible indicator that it is sister to the rest ofMylothris . It is highly probable that we have uncovered all major species groups. The unsampled species are all, as far as we can tell, clearly morphologically related to already sampled species. The sampling process was conducted in the context of a revision of the genus (Warren-Gash in press), and large quantities of material were examined and dissected, including many specimens too old to be readily amenable to molecular sequencing. The unsampled species showed no differences which were impor- tant enough in terms of morphology or genitalia to suggest the likely existence of other species groups. Regardless of the relationships of the major species groups, the biogeographic history of the deeper clades appears to be clear. We infer from our analyses that the group originated in East Cen- tral Africa, and from there spread to other parts of Africa. The fragmentation of the forests in the Miocene and Pleistocene may have contributed to the diversification of the group, perhaps by caus- ing populations to be fragmented and isolated from each other. This appears to be a common pattern in forest dwelling butterflies of Africa (Aduse-Poku et al. 2009; van Velzen et al. 2013; Dhungel and Wahlberg 2018), but the mechanisms leading to their diversification are still poorly understood. Within the species groups of Mylothris we find genetic variation between some of the species to be minimal, and indeed some species share the same haplotypes for the markers sequenced in this study. The infra-specific variation withinMylothris species is, however, considerable, and that has been given full weight both in the preparation of this paper and the forthcoming revision. A number of morphs previously considered as species have been synonymised in consequence (Warren-Gash in press). Nonetheless, such close genetic similarity needs explanation if the specific separation of these groups is to be justified. There is no single answer to why some species are genetically identical for the markers we have sequenced. In one case – Mylothris agathina and M. chloris – they are sympatric with no evidence of interbreeding, and in addition the female genitalia of the two are clearly and consistently different. In the M. jacksoni group, the wing patterns vary, as does the biome in which they fly. Many of the scarcer species, both sampled and unsampled, are montane vicariants; others fly sympatrically without interbreeding and in several cases where the early stages have been observed, the larvae are more distinct than the adults and use different larval foodplants (e.g.M. jacksoni and M. sagala). They not only look different but in their foodplant preferences behave differently. In other groups, there are examples of species which fly together in at least a part of their range, which are morphologically similar in one sex (usually the male) but distinctively different in the other (e.g. M. sulphurea basalis Aurivillius, 1906 and M. aurantiaca Rebel, 1914, with overlap- ping ranges in the north-east of the DRC). A group of more than a dozen species in the hilara group, which have in common a red basal patch at the base of the forewing and broadly similar (but not identical) male genitalia, is found across the width of the main forest belt. A few are very local. In the rugged terrain of the mountains west of the Albertine Nota Lepi. 43: 1–14 11

Rift, they are separated more by terrain than geographical distance, but in those few instances where they overlap, they do not interbreed. At lower altitudes it is quite usual for several species in the group to fly together in varying combinations, without any evidence of cross-fertilisation. In the Albertine Rift widely varying female forms are found which occur nowhere else. Morphology shows up what appear to be widely separated vicariants of similar species in east and west Africa, more akin to each other than those flying in between. They almost certainly use different larval foodplants. Some are tied to strictly forest environments (e.g. M. continua); others prefer more open country (e.g. M. rueppellii Koch, 1865) The case of the jaopura complex, with nine named and mostly allopatric species, offers a different challenge. Those sequenced are indistinguishable on that basis, and the genitalia, while distinctive within the complex, are very similar. However, morphology in terms of size, wingshape and markings is distinctive in each case, as very often is the biome in which they occur. Where they overlap (e.g. M. kiwuensis and M. nsp2), they do not interbreed. Again, and given the very different environments in which they occur, it is highly probable that their larval foodplants also differ. What they have in common is that in practically every case they mimic a Mylothris species in another group, sometimes so well that they can only be separated with certainty by dissection or sequencing. Given the factors discussed above, the markers we have used are probably not sufficiently variable to differentiate the closely related species. It is likely that a genomic approach would shed further light on the species boundaries, as has been found in recent studies on nymphalid butterflies (Dinca et al. 2019; Campbell et al. 2020). Our results do however suggest that radiation within these groups is likely to be recent. Similar minimal genetic differentiation is found in the pierid genus Colias Fabricius, 1807 (Wheat and Watt 2008; Laiho and Ståhls 2013), but interestingly not in the closely related genus Delias (Braby et al. 2007; Müller et al. 2013). The reasons behind the close genetic relationships of several groups of species is not entirely clear, but may have to do with the repeated contraction and expansion of the forests in Africa, especially during the Miocene and Pleistocene. Similar patterns appear to be the case in other forest specialist genera of the family Nymphalidae: Charaxes Ochsenheimer, 1816 (Aduse-Poku et al. 2009), Cymothoe Hübner, 1819 (van Velzen et al. 2013) and Euphaedra Hübner, 1819 (Dhungel and Wahlberg 2018). Mylothris and the nymphalid genera cited appear to be going through rapid speciation in African forests and they require further study to understand the driving mechanisms

Acknowledgements We thank Robin Pranter for help in the laboratory. We thank Steve Collins and ABRI, Kenya for providing us with much useful material and helpful insights. We also thank David Lees for exten- sive comments on the manuscript. NW acknowledges funding from the Swedish Research Council (grant number 2015-04441). We thank the Societas Europea Lepidopterologica (SEL) for support in publishing this article.

References Aduse-Poku K, Brakefield PM, Wahlberg N, Brattström O (2017) Expanded molecular phylogeny of the genus Bicyclus shows the importance of increased sampling for detecting semi-cryptic species and highlights potentials for future studies. Systematics and Biodiversity 15: 115–130. https://doi.org/10.1080/1477200 0.2016.1226979 12 Haydon Warren-Gash et al.: Mylothris phylogeny

Aduse-Poku K, Brattström O, Kodandaramaiah U, Lees DC, Brakefield P, Wahlberg N (2015) Systematics and historical biogeography of the Old World butterfly subtribe Mycalesina (Lepidoptera: Nymphalidae: Satyrinae). BMC Evolutionary Biology 15: 167. https://doi.org/10.1186/s12862-015-0449-3 Aduse-Poku K, Vingerhoedt E, Wahlberg N (2009) Out-of-Africa again: a phylogenetic hypothesis of the genus Charaxes (Lepidoptera: Nymphalidae) based on 5 gene regions. Molecular Phylogenetics and Evo- lution 53: 463–478. https://doi.org/10.1016/j.ympev.2009.06.021 Braby MF (2006) Evolution of larval food plant associations in Delias Hübner butterflies (Lepidoptera: Pieri- dae). Entomological Science 9: 383–398. https://doi.org/10.1111/j.1479-8298.2006.00185.x Braby MF, Nishida K (2010) The immature stages, larval food plants and biology of Neotropical mistletoe butterflies (Lepidoptera: Pieridae). II. TheCatasticta group (Pierini: Aporiina). Journal of Natural History 44: 1831–1928. https://doi.org/10.1080/00222931003633227 Braby MF, Pierce NE, Vila R (2007) Phylogeny and historical biogeography of the subtribe Aporiina (Lepi- doptera : Pieridae): implications for the origin of Australian butterflies. Biological Journal of the Linnean Society 90: 413–440. https://doi.org/10.1111/j.1095-8312.2007.00732.x Campbell EO, Gage EV, Gage RV, Sperling FAH (2020) Single nucleotide polymorphism – based species phylogeny of greater fritillary butterflies (Lepidoptera: Nymphalidae: Speyeria) demonstrates widespread mitonuclear discordance. Systematic Entomology 45: in press. https://doi.org/10.1111/syen.12393 Chazot N, Wahlberg N, Freitas AVL, Mitter C, Labandeira C, Sohn J-C, Sahoo RK, Seraphim N, de Jong R, Heikkilä M (2019) Priors and posteriors in Bayesian timing of divergence analyses: the age of butterflies revisited. Systematic Biology 68: 797–813. https://doi.org/10.1093/sysbio/syz002 Chernomor O, von Haeseler A, Minh BQ (2016) Terrace aware data structure for phylogenomic inference from supermatrices. Systematic Biology 65: 997–1008. https://doi.org/10.1093/sysbio/syw037 Dhungel B, Wahlberg N (2018) Molecular systematics of the subfamily Limenitidinae (Lepidoptera: Nymph- alidae). PeerJ 6: e4311. https://doi.org/10.7717/peerj.4311 Dinca V, Lee KM, Vila R, Mutanen M (2019) The conundrum of species delimitation: a genomic perspective on a mitogenetically super-variable butterfly. Proceedings of the Royal Society of London B Biological Sciences 286: 20191311. https://doi.org/10.1098/rspb.2019.1311 Drummond AJ, Suchard MA, Xie D, Rambaut A (2012) Bayesian phylogenetics with BEAUTi and BEAST 1.7. Molecular Biology and Evolution 29: 1969–1973. https://doi.org/10.1093/molbev/mss075 Guindon S, Dufayard J-F, Lefort V, Anisimova M, Hordijk W, Gascuel O (2010) New algorithms and methods to estimate Maximum-Likelihood phylogenies: Assessing the performance of PhyML 3.0. Systematic Biology 59: 307–321. https://doi.org/10.1093/sysbio/syq010 Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Win- dows 95/98/NT. Nucl. Acids. Symp. Ser. 41: 95–98. Hoang DT, Chernomor O, von Haeseler A, Minh BQ, Vinh LS (2018) UFBoot2: Improving the ultrafast bootstrap approximation. Molecular Biology and Evolution 35: 518–522. https://doi.org/10.1093/molbev/ msx281 Kalyaanamoorthy S, Minh BQ, Wong TKF, von Haeseler A, Jermiin LS (2017) ModelFinder: Fast model selection for accurate phylogenetic estimates. Nature Methods 14: 587–589. https://doi.org/10.1038/ nmeth.4285 Kodandaramaiah U, Wahlberg N (2007) Out-of-Africa origin and dispersal mediated diversification of the butterfly genus Junonia (Nymphalidae: Nymphalinae). Journal of Evolutionary Biology 20: 2181–2191. https://doi.org/10.1111/j.1420-9101.2007.01425.x Laiho J, Ståhls G (2013) DNA barcodes identify Central Asian Colias butterflies (Lepidoptera, Pieridae). Zookeys 365: 175–196. https://doi.org/10.3897/zookeys.365.5879 Matzke NJ (2014) Model selection in historical biogeography reveals that founder-event speciation is a cru- cial process in island clades. Systematic Biology 63: 951–970. https://doi.org/10.1093/sysbio/syu056 Nota Lepi. 43: 1–14 13

Miller MA, Pfeiffer W, Schwartz T (2010) Creating the CIPRES Science Gateway for inference of large phylogenetic trees. Proceedings of the Gateway Computing Environments Workshop (GCE), New Orleans, Louisiana. https://doi.org/10.1109/GCE.2010.5676129 Mitter KT, Larsen TB, de Prins W, De Prins J, Collins S, Weghe GV, Safian S, Zakharov EV, Hawthorne DJ, Kawahara AY, Regier JC (2011) The butterfly subfamily Pseudopontiinae is not monobasic: marked genetic diversity and morphology reveal three new species of Pseudopontia (Lepidoptera: Pieridae). Systematic Entomology 36: 139–163. https://doi.org/10.1111/j.1365-3113.2010.00549.x Müller CJ, Matos-Maraví PF, Beheregaray LB (2013) Delving into Delias Hübner (Lepidoptera: Pieridae): fine-scale biogeography, phylogenetics and systematics of the world’s largest butterfly genus. Journal of Biogeography 40: 881–893. https://doi.org/10.1111/jbi.12040 Nazari V, Larsen TB, Lees DC, Brattström O, Bouyer T, Van de Poel G, Hebert PDN (2011) Phylogenetic systematics of Colotis and associated genera (Lepidoptera: Pieridae): evolutionary and taxonomic implications. Journal of Zoological Systematics and Evolutionary Research 49: 204–215. https://doi.org/10.1111/ j.1439-0469.2011.00620.x Nguyen L-T, Schmidt HA, von Haeseler A, Minh BQ (2015) IQ-TREE: A fast and effective stochastic algo- rithm for estimating maximum likelihood phylogenies. Molecular Biology and Evolution 32: 268–274. https://doi.org/10.1093/molbev/msu300 Peña C, Malm T (2012) VoSeq: a Voucher and DNA Sequence Web Application. PlosOne 7: e39071. https:// doi.org/10.1371/journal.pone.0039071 Price BW, Villet MH, Walton SM, Barker NP (2011) Using molecules and morphology to infer the phylogenetic relationships and evolutionary history of the Dirini (Nymphalidae: Satyrinae), a tribe of butterflies endemic to Southern Africa. Systematic Entomology 36: 300–316. https://doi.org/10.1111/j.1365- 3113.2010.00560.x Rambaut A, Suchard MA, Xie D, Drummond AJ (2014) Tracer v1.6. https://github.com/beast-dev/tracer/releases Ree RH, Moore BR, Webb CO, Donoghue MJ (2005) A likelihood framework for inferring the evolution of geographic range on phylogenetic trees. Evolution 59: 229–2311. https://doi.org/10.1111/j.0014-3820.2005. tb00940.x Ree RH, Sanmartín I (2018) Conceptual and statistical problems with the DEC+J model of founder-event speciation and its comparison with DEC via model selection. Journal of Biogeography in press. https:// doi.org/10.1111/jbi.13173 Ree RH, Smith SA (2008) Maximum Likelihood inference of geographic range evolution by dispersal, local extinction, and cladogenesis. Systematic Biology 57: 4–14. https://doi.org/10.1080/10635150701883881 Sahoo RK, Lohman DJ, Wahlberg N, Müller CJ, Brattström O, Collins SC, Peggie D, Aduse-Poku K, Ko- dandaramaiah U (2018) Evolution of Hypolimnas butterflies (Nymphalidae): Out-of-Africa origin and Wolbachia-mediated introgression. Molecular Phylogenetics and Evolution 123: 50–58. https://doi. org/10.1016/j.ympev.2018.02.001 Silva Brandão KL, Wahlberg N, Francini RB, Azeredo-Espin AML, Brown Jr. KS, Paluch M, Lees DC, Fre- itas AVL (2008) Phylogenetic relationships of butterflies of the tribe Acraeini (Lepidoptera, Nymphalidae, Heliconiinae) and the evolution of host plant use. Molecular Phylogenetics and Evolution 46: 515–531. https://doi.org/10.1016/j.ympev.2007.11.024 Toussaint EFA, Vila R, Yago M, Chiba H, Warren AD, Aduse-Poku K, Storer C, Dexter KM, Maruyama K, Lohman DJ, Kawahara AY (2019) Out of the Orient: Post-Tethyan transoceanic and trans-Arabian routes fostered the spread of Baorini skippers in the Afrotropics. Systematic Entomology 44: 926–938. https:// doi.org/10.1111/syen.12365 van Velzen R, Wahlberg N, Sosef MSM, Bakker FT (2013) Effects of changing climate and host plant associ- ation on species diversification rates in Cymothoe (Lepidoptera, Nymphalidae) tropical forest butterflies. Biological Journal of the Linnean Society 108: 546–564. https://doi.org/10.1111/bij.12012 14 Haydon Warren-Gash et al.: Mylothris phylogeny

Wahlberg N, Rota J, Braby MF, Pierce NE, Wheat CW (2014) Revised systematics and higher classification of pierid butterflies (Lepidoptera: Pieridae) based on molecular data. Zoologica Scripta 43: 641–650.https:// doi.org/10.1111/zsc.12075 Wahlberg N, Wheat CW (2008) Genomic outposts serve the phylogenomic pioneers: designing novel nuclear markers for genomic DNA extractions of Lepidoptera. Systematic Biology 57: 231–242. https://doi. org/10.1080/10635150802033006 Warren-Gash H (in press) Revision of the genus Mylothris. African Natural History Research Trust, London, UK. Wheat CW, Watt WB (2008) A mitochondrial-DNA-based phylogeny for some evolutionary-genetic model species of Colias butterflies (Lepidoptera, Pieridae). Molecular Phylogenetics and Evolution 47: 893–902. https://doi.org/10.1016/j.ympev.2008.03.013

Supplementary material 1 Table S1 Authors: Haydon Warren-Gash, Kwaku Aduse-Poku, Leidys Murillo-Ramos, Niklas Wahlberg Data type: List of specimens used in the study along with NCBI accession numbers. Explanation note: List of specimens used in this study, with species designations, NCBI accession numbers and collection localities. An “X” instead of an accession number indicates a sequence less than 200 bp (and thus unacceptable by NCBI). Copyright notice: This dataset is made available under the Open Database License (http://open- datacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agree- ment intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/nl.43.46354.suppl1

Supplementary material 2 Table S2 Authors: Haydon Warren-Gash, Kwaku Aduse-Poku, Leidys Murillo-Ramos, Niklas Wahlberg Data type: Scaler matrices used in BioGeoBEARS analyses. Explanation note: Scaler matrices used in BioGeoBEARS analyses. Copyright notice: This dataset is made available under the Open Database License (http://open- datacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agree- ment intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/nl.43.46354.suppl2