Zoologica Scripta Revised systematics and higher classification of pierid butterflies (Lepidoptera: Pieridae) based on molecular data NIKLAS WAHLBERG,JADRANKA ROTA,MICHAEL F. BRABY,NAOMI E. PIERCE & CHRISTOPHER W. WHEAT Submitted: 5 May 2014 Wahlberg, N., Rota, J., Braby, M.F., Pierce, N.E. & Wheat, C.W. (2014). Revised Accepted: 12 July 2014 systematics and higher classification of pierid butterflies (Lepidoptera: Pieridae) based on doi:10.1111/zsc.12075 molecular data. — Zoologica Scripta, 43, 641–650. The butterfly family Pieridae comprises approximately 1000 described species placed in 85 genera, but the higher classification has not yet been settled. We used molecular data from eight gene regions (one mitochondrial and seven nuclear protein-coding genes) com- prising a total of ~6700 bp from 96 taxa to infer a well-supported phylogenetic hypothesis for the family. Based on this hypothesis, we revise the higher classification for all pierid genera. We resurrect the tribe Teracolini stat. rev. in the subfamily Pierinae to include the genera Teracolus, Pinacopteryx, Gideona, Ixias, Eronia, Colotis and most likely Calopieris. We transfer Hebomoia to the tribe Anthocharidini and assign the previously unplaced gen- era Belenois and Dixeia to the subtribe Aporiina. Three lineages near the base of Pierinae (Leptosia, Elodina and Nepheronia + Pareronia) remain unplaced. For each of these, we describe and delineate new tribes: Elodinini Braby tribus nova, Leptosiaini Braby tribus nova and Nepheroniini Braby tribus nova. The proposed higher classification is based on well-supported monophyletic groups and is likely to remain stable even with the addition of more data. Corresponding author: Niklas Wahlberg, Department of Biology, University of Turku, Turku, 20014, Finland. E-mail: niklas.wahlberg@utu.fi Niklas Wahlberg, and Jadranka Rota, Department of Biology, University of Turku, Turku, 20014, Finland. E-mail: niklas.wahlberg@utu.fi, jadranka.rota@utu.fi Michael F. Braby, Department of Land Resource Management, PO Box 496, Palmerston, NT 0831, Australia and Research School of Biology, The Australian National University, Can- berra, ACT 0200, Australia. E-mail: [email protected] Naomi E. Pierce, Museum of Comparative Zoology, Harvard University, 26 Oxford Street, Cambridge MA 02138, USA. E-mail: [email protected] Christopher W. Wheat, Department of Zoology, Stockholm University, 10691 Stockholm, Sweden. E-mail: [email protected] Introduction Heikkil€a et al. 2012), but relationships among the four The butterfly family Pieridae is a relatively small family of subfamilies and several genera within the subfamilies have Lepidoptera comprising about 1000 species placed in four remained uncertain. subfamilies and 85 genera. Species of Pieridae have long The most comprehensive study to date (Braby et al. been the subjects of ecological and evolutionary studies 2006) sequenced one nuclear gene region from representa- (e.g. Watt et al. 1977; Wiklund et al. 1991, 1996; Stavenga tives of 74 genera and for a subset of 30 of these, an addi- et al. 2004, 2006; Kemp et al. 2005; Braby 2006; Braby & tional two nuclear gene regions and one mitochondrial Trueman 2006; Wheat et al. 2007; Braby & Nishida 2010; gene region. They found that the four subfamilies were Dinca et al. 2013), but the phylogenetic relationships of the well-supported monophyletic groups, and their results sug- major lineages within the family have only recently been gested that Pseudopontiinae were sister to Dismorphiinae, examined in detail (Braby et al. 2006). The monophyly of and Coliadinae were sister to Pierinae. The position of the family is well established (Wahlberg et al. 2005; Pseudopontiinae has, however, not been stable; for exam- ª 2014 Royal Swedish Academy of Sciences, 43, 6, November 2014, pp 641–650 641 Pieridae systematics N. Wahlberg et al. ple, Heikkil€a et al. (2012) found, with much lower taxon wingless. PCR and sequencing protocols followed Wahlberg sampling, that Pseudopontiinae were sister to Pierinae & Wheat (2008). Alignment of gene regions was straight- based on one mitochondrial and seven nuclear gene forward, as all are protein-coding genes with a conserved regions. Within Pierinae, Braby et al. (2006) found that the codon structure. These gene regions have been used tribes Anthocharidini and Pierini were not monophyletic, successfully for studies of other butterfly relationships and thus, they removed a number of genera from these (Wahlberg et al. 2009; Heikkil€a et al. 2012). Sequences two tribes. Some of these genera were left incertae sedis. were managed, and data sets created using VoSeq (Pena~ & Within Pieridae, several studies have been published Malm 2012). investigating relationships of species at the genus level (Chew Partitioning of large data sets is necessary, especially when & Watt 2006; Braby & Pierce 2007; Braby et al. 2007; different gene regions with different mutational dynamics Wheat & Watt 2008; Mitter et al. 2011; Nazari et al. 2011; are being used. Traditionally, data sets are partitioned by Muller€ et al. 2013). These studies collectively showed that gene region, sometimes divided into codon positions (Rota relationships among supposedly closely related taxa are not 2011). Recently, a new method of partitioning data by rela- that clear and that much additional work remains to be done. tive rates of evolution has been advocated (Cummins & Despite considerable progress in the higher-level system- McInerney 2011; Rota & Wahlberg 2012), and here, we atics of Pieridae, Braby et al. (2006) concluded that many compare the two strategies using Bayes factors – a Bayes fac- issues still require attention. They recommended that tor above 10 is considered as significant (Kass & Raftery future systematic studies of Pieridae concentrate in the fol- 1995; Fan et al. 2011; Xie et al. 2011). The data were first lowing areas: (i) among the higher taxa of Pieridae, deep- partitioned by GENE, using one subdivision for each gene level relationships are still poorly resolved, especially the for a total of eight. The second strategy followed that of relationships among subfamilies; (ii) within the subfamily Rota & Wahlberg (2012), in which the data were sorted Pierinae, relationships of the four major lineages are not according to relative rates of evolution regardless of gene well understood, particularly the informal Colotis group and origin, as calculated by the program TIGER (Cummins & the genus Leptosia, which may prove to constitute separate McInerney 2011). The program was set to subdivide all sites tribes, and further investigation is required to establish into 30 bins of equal ranges of relative rates. Each bin had monophyly and their relationships; and (iii) within the tribe the following number of sites: bin 1 = 3173, bins 2 to Pierini, relationships of the five major lineages are also 12 = 0, bin 13 = 3, bin 14 = 1, bin 15 = 0, bin 16 = 50, bin poorly resolved, particularly the phylogenetic positions of 17 = 3, bin 18 = 21, bin 19 = 9, bin 20 = 15, bin 21 = 53, the genera Elodina, Dixeia and Belenois. Some of these issues bin 22 = 39, bin 23 = 174, bin 24 = 137, bin 25 = 169, bin may only be resolved by inclusion of data from other gene 26 = 352, bin 27 = 474, bin 28 = 544, bin 29 = 1056 and regions that are able to recover deeper level splits. Here, bin 30 = 488. Bins 2 through 23, each of which had fewer we use a large molecular data set comprising one mito- than 200 sites assigned to them, were combined with bin 1 chondrial and seven nuclear gene regions for a total of (which consists of invariable sites) into one subset, as were ~6700 base pairs to investigate the relationships of higher bins 24 and 25. As a result, there were seven partitions in taxa in the family Pieridae. the TIGER-partitioned data. The data were analysed using Bayesian inference and Material and methods maximum likelihood. The maximum-likelihood analyses A total of 96 taxa of Pieridae were sampled for this study were run with RAXML (Stamatakis et al. 2008) with 1000 as well as 14 outgroup taxa from the families Nymphalidae, rapid bootstrap replicates and a search for the maximum- Lycaenidae and Riodinidae taken from a previously pub- likelihood topology on the CIPRES portal. The data were lished study (Heikkil€a et al. 2012). The majority of speci- modelled according to the GTR+G model for each parti- mens sequenced here are the same individuals as those tion independently. Bayesian analyses were conducted with used in a previous study (Braby et al. 2006), with a few MRBAYES 3.2 (Ronquist et al. 2012). The analyses were run specimens collected specifically for this study, and a few for 10 million generations sampling every 1000 genera- taxa for which sequence data were downloaded from NCBI tions, with the number of independent runs depending on (Table S1). Eight gene regions were sequenced for the pie- the analysis (minimally two independent runs). The con- rid samples, including the mitochondrial gene region cyto- vergence of the likelihood traces of the independent runs chrome oxidase subunit I (COI) and the nuclear gene regions was assessed with TRACER v1.5, and the ESS (effective elongation factor-1a (EF-1a), ribosomal protein S5 (RpS5), car- sample size) values were verified to be above 200 for all bamoylphosphate synthase domain protein (CAD), cytosolic parameters, which indicates that all parameters were malate dehydrogenase (MDH), glyceraldehyde-3-phosphate dehy- sufficiently sampled to estimate their posterior distributions drogenase (GAPDH), isocitrate dehydrogenase (IDH) and (Drummond et al. 2006). 642 ª 2014 Royal Swedish Academy of Sciences, 43, 6, November 2014, pp 641–650 N. Wahlberg et al. Pieridae systematics Analyses in MRBAYES 3.2 were run twice independently which we have designated as the Eurema-clade and the Co- according to the parameters specified above for both the lias-clade. GENE-partitioned data and the TIGER-partitioned data. Relationships within Pierinae were somewhat more com- The model-jumping feature of the program was utilised, plex (Fig.