A new sectional classification of Lachenalia (Asparagaceae) based on a multilocus DNA phylogeny

Lachenalia J.Jacq. ex Murray (Asparagaceae; Scilloideae; Hyacintheae) is a large and morphologically diverse genus of more than 140 bulbous species endemic to southern Africa. Previous attempts to infer a well resolved and robustly supported phylogeny of Lachenalia using Sanger sequencing of candidate loci and/or morphological characters have been largely unsuccessful. Consequently, the current infrageneric classification is artificial and there is a need to explore alternative avenues to produce a phylogenetic classification. In this paper we present a novel phylogenetic hypothesis for Lachenalia inferred using maximum likelihood and coalescent-based species tree estimation (ASTRAL) as applied to 378 hybrid-enrichment loci. Our tree is well resolved and well supported, providing strong support for a monophyletic radiation of the genus in southern Africa and a solid foundation for a revised infrageneric classification. The well-supported placement of L. isopetala Jacq. as sister to Lachenalia + Massonia supports the establishment of a new monotypic genus, Pseudolachenalia G.D.Duncan, to accommodate this species. Conversely, the inclusion of species previously classified as Polyxena Kunth within the Lachenalia clade supports the transfer of these species to Lachenalia. Within Lachenalia, the delimitation of subgenera and sections is complicated by the highly imbalanced character of the phylogeny and by the high levels of homoplasy shown by most morphological characters traditionally used to delimit species in this group. Nonetheless, we propose an infrageneric taxonomy comprising 10 morphologically distinct, monophyletic sections. The largest of these, section Lachenalia, is further divided into 13 more-or-less diagnosable, monophyletic subsections. Keys to the sections of Lachenalia, and to the subsections of section Lachenalia, are provided.Funding provided by: National Research Foundation South Africa*Crossref Funder Registry ID: Award Number: 105976Sampling Actively growing leaf material of 132 Lachenalia accessions, representing 118 species plus a further five subspecies, were sampled from the wild-provenance collection of indigenous bulbous taxa in cultivation in the bulb nursery at Kirstenbosch National Botanical Garden (Table 1). The balance of the Lachenalia samples were replicate collections of selected widespread and/or morphologically variable species. In addition, we sampled, as outgroups, Massonia bifolia (Jacq.) J.C.Manning & Goldblatt, M. depressa Houtt., M. pustulata Jacq. and Veltheimia capensis (L.) DC, giving a total of 136 accessions. Voucher specimens of sampled accessions are lodged in the Compton Herbarium (NBG). DNA isolation and sequencing DNA isolation from plant tissue was done according to the protocol of Healey & al. (2014). Two grams of fresh leaf material were frozen and pulverized in liquid nitrogen using a mortar and pestle. As recommended by Healey & al. (2014), all steps were performed in 50 ml Falcon tubes, with the final DNA pellets eluted in 200 µl of Tris-EDTA (TE) buffer. Where pellets did not resuspend fully, an additional amount (typically 100-200 µl) of TE buffer was added until the pellet was fully dissolved. The DNA samples were then purified using the Qiagen Genomic DNEasy Clean and Concentrator Kit (Qiagen N.V., Venlo, Nederland) according to the manufacturer's instructions. Thereafter, the quality and quantity of DNA were assessed, both by running and visualizing samples in a 1% TBE agarose gel, containing ethidium bromide, and using a NanoDrop spectrophotometer. Sequence data collection DNA sequences were collected in collaboration with the Center for Anchored Phylogenomics (www.anchoredphylogeny.com). Libraries were prepared following a protocol originally developed by Meyer & Kircher (2010) and later modified by Prum & al. (2015). Extracted DNA was quantified using a Qubit fluorometer (ThermoFisher Scientific), then sonicated to a size distribution of 250-400 bp using a Covaris ultrasonicator. Following end repair, common Illumina adapters were ligated onto the blunt ends, after which sample-specific 8 bp single indexes were introduced using an indexing PCR step. Libraries were pooled in equal concentration in three batches of ca.16 samples, prior to enrichment using the AHE Angiosperm kit (v.1) described in Buddenhagen & al. (2016), Mitchell & al. (2017) and Léveillé-Bourret & al. (2018). This kit targets 517 AHE loci. The AHE Angiosperm kit was derived from 25 genomes from species across the Angiosperm clade. The targets for this kit are exons from genes that are low-copy and align well across the angiosperms due to their intermediate levels of variation. These loci were not selected based on function. Although these target exons are somewhat short (each typically